Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof

ABSTRACT

This invention relates to amylase polypeptides, and nucleic acids encoding the polpypeptides and uses thereof. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the current invention show an altered exospecifity.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of and priority to U.S. Ser. No.60/485,616, entitled “Exo-specific Amylase Polypeptides, Nucleic AcidsEncoding Those Polypeptides and Uses Thereof” (Docket No. GC807P), filedJul. 7, 2003, by Berg, et al, and U.S. Ser. No. 60/485,413, entitled,“Thermostable Amylase Polypeptides, Nucleic Acids Encoding ThosePolypeptides and Uses Thereof (Docket No. GC806P), filed Jul. 7, 2003.These applications are related to U.S. Ser. No. 60/485,539, entitled“Polypeptides”, filed Jul. 7, 2003 (Docket number P016939USO).

FIELD OF THE INVENTION

This invention relates to amylase polypeptides, and nucleic acidsencoding the polpypeptides and uses thereof. The amylases of the presentinvention have been engineered to have more beneficial qualities.Specifically, the amylases of the current invention show an alteredexospecifity.

BACKGROUND OF THE INVENTION

Improved amylases can ameliorate problems inherent in certain processes,such as baking. Crystallisation of amylopectin takes place in starchgranules days after baking, which leads to increased firmness of breadand causes bread staling. When bread stales, bread loses crumb softnessand crumb moisture. As a result, crumbs become less elastic, and breaddevelops a leathery crust.

Enzymatic hydrolysis (by amylases, for example) of amylopectin sidechains can reduce crystallization and increase anti-staling.Crystallization depends upon the length of amylopectin side chains: thelonger the side chains, the greater the crystallization. Most starchgranules are composed of a mixture of two polymers: amylopectinand-amylose, of which about 75% is amylopectin. Amylopectin is a verylarge, branched molecule consisting of chains of α-D-glucopyranosylunits joined by (1-4) linkages, where the chains are attached byα-D-(1-6) linkages to form branches. Amylose is a linear chain of (1-4)linked α-D-glucopyranosyl units having few α-D-(1-6) branches.

Baking of farinaceous bread products such as white bread, bread madefrom bolted rye flour and wheat flour and rolls is accomplished bybaking the bread dough at oven temperatures in the range of from 180 to250° C. for about 15 to 60 minutes. During the baking process a steeptemperature gradient (200→120° C.) prevails over the outer dough layerswhere the crust of the baked product is developed. However, due tosteam, the temperature in the crumb is only about 100° C. at the end ofthe baking process. Above temperatures of about 85° C., enzymeinactivation can take place and the enzyme will have no anti-stalingproperties. Only thermostable amylases, thus, are able to modify starchefficiently during baking.

Endoamylase activity can negatively affect the quality of the finalbread product by producing a sticky or gummy crumb due to theaccumulation of branched dextrins. Exoamylase activity is preferred,because it accomplishes the desired modification of starch that, leadsto retardation of staling, with fewer of the negative effects associatedwith endoamylase activity. Reduction of endoamylase activity can lead togreater exospecifity, which can reduce branched dextrins and produce ahigher quality bread.

The present invention is drawn to polypeptides which have alteredexospecificity.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a polypeptide comprising a PS4variant, the PS4 variant being derivable from a parent polypeptide. Theparent enzyme may preferably be a Pseudomonas saccharophilanon-maltogenic exoamylase, such as the exoamlyase having the amino acidsequence set forth in SEQ ID NO:1 or SEQ ID NO:5. The parent enzyme maypreferably be a Pseudomonas stutzeri non-maltogenic exoamylase, such asa polypeptide having the amino acid sequence set forth in SEQ ID NO: 7or SEQ ID NO11. Other members of the PS4 family may be used as parentenzymes.

In preferred embodiments, the parent polypeptide is a non-maltogenicexoamylase from Pseudomonas saccharophilia having the amino acidsequence set forth in SEQ ID NO: 1 or set forth in SEQ ID NO:5. In otherpreferred embodiments, the parent polypeptide is a non-maltogenicexoamylase from Pseudomonas stutzeri having the amino acid sequence setforth in SEQ ID NO:7 or set forth in SEQ ID NO:11. In a preferredembodiment, the PS4 variant differs from the parent polypeptide byincluding amino acid substitutions, the substitutions located at aposition comprising at least one position selected from the groupconsisting of: 4, 9, 13, 33, 34, 42, 70, 71, 87, 99, 100, 108, 113, 121,131, 134, 135, 141, 153, 157, 158, 160, 161, 166, 170, 171, 178, 179,184, 188, 198, 199, 221, 223, 238, 270, 277, 290, 307, 315, 334, 335,342, 343, 372, 392, 398, 399, 405, 415, 425, wherein reference toposition numbering is with respect to the Pseudomonas saccharophiliasequence shown as SEQ ID NO: 1. In a preferred embodiment, the PS4variant differs from the parent polypeptide by including amino acidsubstitutions, the substitutions located at a position comprising atleast one position selected from the group consisting of: 4, 33, 34, 70,71, 87, 99, 108, 113, 121, 131, 134, 141, 157, 158, 171, 178, 179, 188,198, 199, 223, 290, 307, 315, 334, 343, 399, and 405, wherein referenceto position numbering is with respect to the Pseudomonas saccharophiliasequence shown as SEQ ID NO: 1. Preferably, the position is at least oneposition selected from the group consisting of: 33, 34, 71, 87, 121,134, 141, 157, 178, 179, 223, 307, 334, and 343. Preferably the PS4variant comprises at least one substitution selected from the groupconsisting of N33Y, D34N, K71R, G87S, G121D, G134R, A141P, L178F, A179T,G223A, H307L, S334P, and D343E.

In another embodiment, the exoamylase further comprises at least oneadditional substitution at a position selected from 108, 158, 171 and188. Preferably the PS4 variant comprises at least one substitutionselected from the group consisting of K108R, G158D, Y171S, and G188A.

Preferably, the PS4 variant comprises at least one substitution selectedfrom the group consisting of: G4D, N33Y, D34N, G70D, K71R,G87S, A99V,K108R, V 131I, G121D, G134R, A141P, 1157L, G158D, Y171S, L178F, A179T,G188A, Y198F, Y198L, A199V, G223A, V290I, H307L, I315V, S334P, D343E,S399P, A405F, and A405E. Preferably, the PS4 variant comprises thefollowing substitutions: N33Y, D34N, G134R, A141P, I157L, G223A, H307Land S334P with at least one additional substitution of L178F or A179T.Preferably, the PS4 variant comprises at least one of the followingsubstitutions: N33Y, D34N, I157L, L178F, A179T, G223A or H307L.Preferably, the PS4 variant comprises at least one of the followingsubstitutions: G87S, G134R, A141P, or S334P.

In other preferred embodiments, the PS4 variant polypeptide comprises acombination selected from:

-   G134R, A141P I157L G223A H307L S334P D343E G121D;-   G134R A141P I157L G223A H307L S334P D343E N33Y G112D;-   G134R A141P I157L G223A H307L S334P D343E N33Y;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G87S G121D S214N T375A;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D Y171S G188A N138D;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T G188A;-   G134R, A141P I157L G223A H307L S334P K71R L178F A179T;-   G134R, A141P I157L G223A H307L S334P L178F A179T;-   G134R, A141P I157L G223A H307L S334P N33Y D34N L178F A179T;-   G134R, A141P I157L G223A H307L S334P L178F A179T G87S G121D;-   G134R, A141P I157L G223A H307L S334P L178F A179T G121D;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D E343D;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T Y33N N34D E343D;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D;-   G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D;-   G87S, V113I, G134R, A141P, I157L, Y198F,G223A,V2901, H307L, S334P,    D343E;-   113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   A99V, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, I157L, Y198F, G223A, V290I, H307L, S334P, D343E-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   A199V, D343E, V113I, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P;-   V113I, A141P, I157L, Y198F, G223A, V290I,S334P, D343E;-   V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P, D343E;-   V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P, D343E;-   V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   V113I, A141P, Y198F, G223A, V290I, H307L;-   V113I, A141P, Y198F, G223A, V290I, S334P, D343E;-   V113I, A141 P, Y1 98F, G223A, A268P, V290I, S399P V113I, A141 P,    Y198F, G223A, V290I, S399P;-   V113I, A141 P, Y198W, G223A, V290I;-   V113I, A141P, Y198F, G223A, V290I;-   Y198F, G223A, V290I;-   Y198W, G223A, V290I;-   V113I, A141P, I157L, Y198F, G223A, V290I;-   V113M;-   V113A;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, I315V,    S334P, D343E;-   D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L,    S334P, D343E;-   K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L,    G313G, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A,    V290I, H307L, G313G, S334P, D343E; V113I, G134R, A141P, I157L,    A179V, Y198F, G223A, V290I, H307L, S334P, D343E;-   V113I, G134R, A141P, I157L, I170I, Y198F, G223A, V290I, H307L,    G313G, S334P, D343E V113I, G134R, A141P, I157L, A179V, Y198F, G223A,    V290I, H307L, G313G, S334P, D434E;-   G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E, A405E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E, A405V;-   A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   V113I, G134R, A141P, I157L, Y198L,G223A, V290I, H307L, S334P, D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P,D343E;-   K108R, V113I, G134R, A141P, I157L, Y198F,G223A, V290I, H307L, S334P,    D343E;-   D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    and-   A141P, G134R, G223A, H307L, I157L, V113I, V2901I, Y198F, G188A;

In other preferred embodiments, the PS4 variant polypeptide comprises acombination selected from:

-   G134R, A141P, I157L, G223A, H307L and S334P;-   G121D, G134R, A141P, I157L, G223A, H307L and S334;-   G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P;-   G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;-   G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;-   G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;-   N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P; N33Y,-   D34N, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;; and-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P.

In other preferred embodiments, the PS4 variant polypeptide comprises acombination selected from the following:

-   N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P; and-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P

In preferred embodiments, the PS4 variant is an amino acid comprisingthe sequence set forth in either SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4;SEQ ID NO 4a, SEQ ID NO 4b, SEQ ID NO 4c, SEQ ID NO:8, SEQ ID NO:9 orSEQ ID NO:10.

The PS4 variant can be derived from a Pseudomonas sp. In an embodiment,the Pseudomonas species is selected from Pseudomonas saccharophilia andPseudomonas stutzeri.

The PS4 variant polypeptide may comprise one or more mutations inaddition to 5 those set out above. Other mutations, such as deletions,insertions, substitutions, transversions, transitions and inversions, atone or more other locations, may also be included. Likewise, thepolypeptide may be missing at least one of the substitutions set forthabove.

In a preferred embodiment, the polypeptide is truncated. The truncationmay be at 10 the N-terminal end or the C-terminal end. The parent enzymeor PS4 variant may lack one or more portions, such as sub-sequences,signal sequences, domains or moieties, whether active or not. Forexample, the parent enzyme or the PS4 variant polypeptide may lack asignal sequence, as described herein. Alternatively, or in addition, theparent enzyme or the PS4 variant may lack one or more catalytic orbinding domains. In preferred embodiments, is the parent enzyme or PS4variant may lack one or more of the domains present in non-maltogenicexoamylases, such as the starch binding domain. For example, the PS4polypeptides may have only sequence up to position 429, relative to thenumbering of a Pseudomonas saccharophilia non-maltogenic exoamylaseshown as SEQ ID NO: 1. In a preferred embodiment, the PS4 variantspSac-d34 (SEQ ID NO 4c, FIG. 8 c), pSac-D20 (SEQ ID NO 4a, FIG. 8 a) andpSac-D14 (SEQ ID NO 4b, FIG. 8 b) are provided, the variants having anamino acids sequences as set forth in the Figures.

The PS4 variant may also comprise a homologous sequence. A homologoussequence comprises a nucleotide sequence at least 75, 80, 85 or 90%identical, preferably at least 95, 96, 97, 98 or 99% identical to anucleotide sequence encoding a PS4 variant polypeptide enzyme.

Preferred embodiments also include functional equivalents. The PS4variant polypeptides described in this document are derived from, or arevariants of, polypeptides preferably exhibiting non-maltogenicexoamylase activity. Preferably, the parent enzymes are non-maltogenicexoamylases themselves. The PS4 variant polypeptides in preferredembodiments also exhibit non-maltogenic exoamylase activity.

The PS4 variants described herein will preferably-have exospecificity,for example measured by exo-specificity indices, as described herein,consistent with their being exoamylases. Moreover, they preferably havehigher or increased exospecificity when compared to the parent enzymesor polypeptides from which they are derived. Thus, for example, the PS4variant polypeptides may have an exo-specificity index of 20 or more,i.e., its total amylase activity (including exo-amylase activity) is 20times or more greater than its endoamylase activity. In preferredembodiments, the exo-specificity index of exoamylases is 30 or more, 40or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or100 or more. In preferred embodiments, the exo-specificity index is 150or more, 200 or more, 300 or more, or 400 or more.

Preferably, the PS4 variant will be more thermostable than the-parent.Preferably, the PS4 variant polypeptide is capable of degrading starchat temperatures of from about 55° C. to about 80° C. or more.Preferably, the PS4 variant retains its activity after exposure totemperatures of up to about 95° C. The PS4 variant polypeptidesdescribed here have half lives extended relative to the parent enzyme bypreferably 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% ormore, preferably at elevated temperatures of from 55° C. to about 95° C.or more, preferably at about 80° C. Preferably, the sample is heated for1-10 minutes at 80° C. or higher.

Preferably, the PS4 variant polypeptide is more pH stable. Preferably,it has a higher pH stability than its cognate parent polypeptide.Preferably, the PS4 variant polypeptide is capable of degrading starchat a pH of from about 5 to about 10.5. The PS4 variant polypeptides mayhave 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longerhalf life when compared to their parent polypeptides under identical pHconditions. In another embodiment, the degree of pH stability may beassayed by measuring the activity or specific activity of the enzyme inspecific pH conditions. The specific pH conditions may be any pH frompH5 to pH10.5.

In preferred embodiments, the functional equivalents will have sequencehomology to at least one of the PS4 family members. Functionalequivalents will have sequence homology to either of the Pseudomonassaccharophila and Pseudomonas stutzeri non-maltogenic exoamylasesmentioned above, preferably both. The functional equivalent may alsohave sequence homology with any of the sequences set out as SEQ ID NOs:1 to 12, preferably SEQ ID NO: 1 or SEQ ID NO: 7 or both. Sequencehomology is preferably at least 60%, preferably 65% or more, preferably75% or more, preferably 80% or more, preferably 85% or more, preferably90% or more, preferably 95% or more. Sequence homologies may begenerated by any or all of the programs set forth herein. In otherembodiments, the functional equivalents will be capable of specificallyhybridising to any of the sequences set out above.

In a second aspect, the invention provides a nucleic acid, the nucleicacid encoding a polypeptide comprising a PS4 variant being derivablefrom a parent polypeptide, as set forth above. The parent enzyme maypreferably be a Pseudomonas saccharophila non-maltogenic exoamylase asset forth in SEQ ID NO:1 or as set forth in SEQ ID NO:5. The parentenzyme may preferably be a Pseudomonas stutzeri non-maltogenicexoamylase, as set forth in SEQ ID NO: 7 or as set forth in SEQ ID NO11.Other members of the PS4 family may be used as parent enzymes.

In preferred embodiments, the parent polypeptide is a non-maltogenicexoamylase from Pseudomonas saccharophilia non-maltogenic exoamylasehaving a sequence as set forth in SEQ ID NO: 1 or as set forth in SEQ IDNO:5. In other preferred embodiments, the parent polypeptide comprises anon-maltogenic exoamylase from Pseudomonas stutzeri having a sequenceshown as set forth in SEQ ID NO:7 or as set forth in SEQ ID NO:11. In apreferred embodiment, the nucleic acid encoding the PS4 variant differsfrom the parent nucleic acid by encoding amino acid substitutions, thesubstitutions located at a position comprising at least one positionselected from the group consisting of: 4, 9, 13, 33, 34, 42, 70, 71, 87,99, 100, 108, 113, 121, 131, 134, 135, 141, 153, 157, 158, 160, 161,166, 170, 171, 178, 179, 184, 188, 198, 199, 221, 223, 238, 270, 277,290,307, 315, 334, 335, 342, 343, 372, 392, 398, 399, 405, 415, 425,wherein reference to position numbering is with respect to a Pseudomonassaccharophilia sequence set forth in SEQ ID NO: 1. Preferably, thepositions are at least one position selected from the group consistingof: 33, 34, 87, 121, 134, 141, 157, 178, 179, 223, 307 and 334.

Preferably, the nucleic acid encoding the PS4 variant comprises anucleic acid encoding at least one substitution in the polypeptide, thesubstitution selected from the group consisting of: N33Y, D34N, G87S,G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P.Preferably, the nucleic acid encoding the PS4 variant comprises thefollowing substitutions: N33Y, D34N, G134R, A141P, I157L, G223A, H307Land S334P with at least one additional substitution of L178F or A179T.Preferably, the nucleic acid encoding the PS4 variant comprises one ofthe following substitutions: N33Y, D34N, I157L, L178F, A179T, G223A orH307L. Preferably, the nucleic acid encoding the PS4 variant comprisesone of the following substitutions: G87S, G134R, A141P, or S334P. Inanother embodiment, the nucleic acid encoding the PS4 variant comprisesa nucleic acid encoding at least one of the following substitutions:K71R, K108R, G158D, Y171S, G188A, and D343E.

In another embodiment, the nucleic acid encoding the PS4 variantcomprises a nucleic acid encoding at least one of the followingcombinations: G134R, A141P I157L

-   G223A H307L S334P D343E G121D;-   G134R A141P I157L G223A H307L S334P D343E N33Y G121D;-   G134R A141P I157L G223A H307L S334P D343E N33Y;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G87S G121D S214N T375A;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D Y171S G188A N138D;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T G188A;-   G134R, A141P I157L G223A H307L S334P K71R L178F A179T;-   G134R, A141P I157L G223A H307L S334P L178F A179T;-   G134R, A141P I157L G223A H307L S334P N33Y D34N L178F A179T;-   G134R, A141P I157L G223A H307L S334P L178F A179T G87S G121D;-   G134R, A141P I157L G223A H307L S334P L178F A179T G121D;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D E343D;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T Y33N N34D E343D;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D;-   G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D;-   G87S, V113I, G134R, A141P, I157L, Y198F,G223A,V290I, H307L, S334P,    D343E;-   113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   A99V, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, I157L, Y198F, G223A, V290I, H307L, S334P, D343E-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   A199V, D343E, V 113I, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P;-   V113I, A141P, I157L, Y198F, G223A, V290I, S334P, D343E;-   V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P, D343E;-   V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P, D343E;-   V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   V113I, A141P, Y198F, G223A, V290I, H307L;-   V113I, A141P, Y198F, G223A, V290I, S334P, D343E; V113I, A141P,    Y198F, G223A, A268P, V290I, S399P-   V113I, A141P, Y198F, G223A, V290I, S399P;-   V113I, A141P, Y198W, G223A, V290I;-   V113I, A141P, Y198F, G223A, V290I;-   Y198F, G223A, V290I;-   Y198W, G223A, V290I;-   V113I, A141P, I157L, Y198F, G223A, V290I;-   V113M;-   V113A;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, 1315V,    S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I,    H307L, S334P, D343E;-   V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L,    S334P, D343E;-   K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L,    G313G, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A,    V290I, H307L, G313G, S334P, D343E; V113I, G134R, A141P, I157L,    A179V, Y198F, G223A, V290I, H307L, S334P, D343E;-   V113I, G134R, A141P, I157L, I170I, Y198F, G223A, V290I, H307L,    G313G, S334P, D343E V113I, G134R, A141P, I157L, A179V, Y198F, G223A,    V290I, H307L, G313G, S334P, D343E;-   G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E, A405E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E, A405V;-   A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;-   V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P,    D343E;-   V113I, G134R, A141 P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E;-   K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P, D343E;-   D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343-   G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    and-   A141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F, G188A;-   G134R, A141P, I157L, G223A, H307L and S334P;-   G121D, G134R, A141P, I157L, G223A, H307L and 5334;-   G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P;-   G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;-   G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;-   G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;-   N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P; N33Y, D34N, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P;; and-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P;-   N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P; and-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P

In preferred embodiments, the nucleic acid encoding the PS4 variantencodes an amino acid sequence comprising either SEQ ID NO:2; SEQ IDNO:3; SEQ ID NO:4; SEQ ID NO:4a; SEQ ID NO:4b; SEQ ID NO:4c; SEQ IDNO:8, SEQ ID NO:9 or SEQ ID NO:10.

In a preferred embodiment, the nucleic acid encodes a truncatedpolypeptide. The truncation may be at the N-terminal end or theC-terminal end. The parent enzyme or PS4 variant may lack one or moreportions, such as sub-sequences, signal sequences, domains or moieties,whether active or not. For example, the parent enzyme or the PS4 variantpolypeptide may lack a signal sequence. Alternatively, or in addition,the parent enzyme or the PS4 variant may lack one or more catalytic orbinding domains. In a preferred embodiment, the parent enzyme or PS4variant may lack one or more of the domains present in non-maltogenicexoamylases, such as the starch binding domain. For example, the PS4polypeptides may have only sequence up to position 429, relative to thenumbering of a Pseudomonas saccharophilia non-maltogenic exoamylaseshown as SEQ ID NO: 1. In 5 a preferred embodiment, the nucleic acidencodes the PS4 variants pSac-d34, pSac-D20 and pSac-D14 as set forth inthe Figures.

The nucleic acid encoding the PS4 variant polypeptide may comprise oneor more mutations in addition to those set out above. Other mutations,such as deletions, insertions, substitutions, transversions, transitionsand inversions, at one or more other locations, may also be included.Likewise, the polypeptide encoded by the nucleic acid may be missing atleast one of the substitutions set forth above.

The nucleic acid encoding the PS4 variant may also comprise ahomologous, sequence. A homologous sequence comprises a nucleotidesequence at least 75, 80, 85 or 90% identical, preferably at least 95,96, 97, 98 or 99% identical to a nucleotide sequence encoding a PS4variant polypeptide enzyme.

Preferred embodiments also include a nucleic acid encoding a polypeptidewhich is a functional equivalent of a PS4 variant. The nucleic acidsencoding PS4 variant polypeptides described in this document are derivedfrom, or are variants of, nucleic acids which preferably encode anenzyme having non-maltogenic exoamylase activity. Preferably, the parentenzymes encoded by the nucleic acids are non-maltogenic exoamylasesthemselves. The PS4 variant polypeptides encoded by the nucleic acids inpreferred embodiments also exhibit non-maltogenic exoamylase activity.

The PS4 variants encoded by the nucleic acids will preferably haveexospecificity, for example measured by exo-specificity indices, asdescribed herein. Moreover, they preferably have higher or increasedexospecificity when compared to the parent enzymes or polypeptides fromwhich they are derived, preferably under identical conditions. Thus, forexample, the PS4 variant polypeptides may have 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200% or higher exo-specificity index. They mayhave 1.5× or higher, 2× or higher, 5× or higher, 10× or higher, 50× orhigher, 100× or higher, when compared to their parent polypeptides,preferably under identical conditions.

Preferably, the PS4 variant encoded by the nucleic acid will be morethermostable than the parent counterpart. Preferably, the PS4 variantpolypeptide is capable of degrading starch at temperatures of from about55° C. to about 80° C. or more. Preferably, the PS4 variant retains itsactivity after exposure to temperatures of up to about 95° C. The PS4variant polypeptides described here have half lives extended relative tothe parent enzyme by preferably 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200% or more, preferably at elevated temperatures of from 55°C. to about 95° C. or more, preferably at about 80° C. Preferably, thesample is heated for 1-10 minutes at 80° C. or higher.

Preferably, the PS4 variant polypeptide encoded by the nucleic acid ispH stable. Preferably, it has a higher pH stability than its parentpolypeptide. Preferably, the PS4 variant polypeptide is capable ofdegrading starch at a pH of from about 5 to about 10.5. The specific pHconditions may be any pH from pH5 to pH10.5. The PS4 variant polypeptideencoded by the nucleic acid may have a longer half life, or a higheractivity (depending on the assay) when compared to the parentpolypeptide under identical conditions. The PS4 variant polypeptide mayhave 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longerhalf life when compared to their parent polypeptide under identical pHconditions. Alternatively, or in addition, they may have higher activitywhen compared to the parent polypeptide under identical pH conditions.

In a preferred embodiment, the functional equivalents encoded by thenucleic acid will have sequence homology to at least one of the PS4family members. Functional equivalents will have sequence homology toeither of the Pseudomonas saccharophila and Pseudomonas stutzerinon-maltogenic exoamylases mentioned above, preferably both, in apreferred embodiment. The functional equivalent may also have sequencehomology with any of the sequences set out as SEQ ID NOs: 1 to 12,preferably SEQ ID NO: 1 or SEQ ID NO: 7 or both. Sequence homology ispreferably at least 60%, preferably 65% or more, preferably 75% or more,preferably 80% or more, preferably 85% or more, preferably 90% or more,preferably 95% or more.

In other embodiments, a nucleic acid complementary to a nucleic acidencoding any of the PS4 variants set forth herein is provided.Additionally, a nucleic acid capable of hybridising to the complement isprovided. In a preferred embodiment, a nucleic acid encoding thefunctional equivalents will be capable of specifically hybridising toany of the sequences set out above is provided herein, as well as itscomplement.

In a preferred embodiment, the sequence for use in the methods andcompositions described here is a synthetic sequence. It includes, but isnot limited to, sequences made with optimal codon usage for hostorganisms—such as the methylotrophic yeasts Pichia and Hansenula.

A third aspect of the invention provides for compositions comprising atleast one PS4 variant polypeptide and another ingredient. The otheringredient may be an enzyme selected from the group consisting ofoxidoreductases, hydrolases, lipases, esterases, glycosidases, amylases,pullulanases, xylanases, cellulases, hemicellulases, starch degradingenzymes, proteases and lipoxygenases. In a preferred embodiment, thecomposition comprises at least one PS4 variant and a maltogenic amylasefrom Bacillus, as disclosed in WO91/04669. A preferred embodimentcomprises a PS4 variant and flour.

The further enzyme can be added together with any dough ingredientincluding the flour, water or optional other ingredients or additives orthe dough improving composition. The further enzyme can be added beforeor after the flour, water and optionally other ingredients and additivesor the dough improving composition. The further enzyme may be a liquidpreparation or in the form of a dry composition.

A fourth aspect provides vectors comprising a PS4 variant polypeptide,cells comprising a PS4 variant polypeptide and methods of expressing aPS4 variant polypeptide. In a preferred embodiment, the invention isdirected to a recombinant replicable vector with a nucleic acid encodinga PS4 variant polypeptide. The vector may additionally comprise any ofthe elements set forth herein. Another preferred embodiment provides ahost cell comprising a nucleic acid encoding a PS4 variant. The hostcell may be any of the bacterial, fungal or yeast cells set forthherein. In a preferred embodiment, the invention is drawn to a method ofexpression of a PS4 polypeptide, as provided herein.

Further aspects of the invention may be found in the relatedapplications, having attorney docket numbers 674510-2007 and GC806P,which are incorporated by reference, herein, including any drawings,references and Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing thermostability improvement of the PS4variants. PS4cc1 is an expressed control enzyme derived from Pseudomonassaccharophilia, without signal sequence and lacking the starch bindingdomain. Half life in minutes is plotted against temperature in degreesC. for PS4cc1, pSac-D3, pSac-D20 and pSac-D14.

FIG. 2 is a graph showing dosage effect of PSac-D34 in a model doughsystem trial. Solid content of crumb was measured by NMR. Firmnessmeasured by solid content is plotted against days after baking forcontrol, 0.5, 1, 2 ppm of D34.

FIG. 3 is a graph shows the results of a baking trial showing reducedfirmness and firming rate upon adding PSac-D3 and Psac-D14 in a dosageof 1 mg per kg of flour. Firmness measured by hPa is plotted againstdays after baking for control,

FIG. 4 shows a baking trial showing the increased softening effect ofPSac-D3 (G134R, A141P, I157L, G223A, A307L, S334P, K71R, D343E, N33Y,D34N, L178F, A179T) compared to Psac-D3 without N33Y, D34N, K71R, L178F,A179T, which has t_(1/2)-75 of 3,6 in contrast to that of PSac-D3 being9,3 min at 75C.

FIG. 5 shows a PS4 (SEQ ID NO: 1) reference sequence, derived fromPseudomonas saccharophila maltotetrahydrolase amino acid sequence.

FIG. 6 shows the sequence of a PS4 variant (SEQ ID NO: 2); Pseudomonassaccharophila maltotetrahydrolase amino acid sequence with substitutionsG134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F and A179T.

FIG. 7 shows the sequence of a PS4 variant (SEQ ID NO: 3); Pseudomonassaccharophila maltotetrahydrolase amino acid sequence with substitutionsG134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F, A179T andG121D.

FIG. 8A shows the sequence of PS4 variant (SEQ ID NO: 4); Pseudomonassaccharophila maltotetrahydrolase amino acid sequence with substitutionsG134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F, A179T,G121D and G87S.

FIG. 8B shows the sequence of PSac-D20 sequence (SEQ ID NO 4a);Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with13 substitutions and deletion of the starch binding domain.

FIG. 8C shows the sequence of PSac-D14 sequence.(SEQ ID NO 4b);Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with14 substitutions and deletion of the starch binding domain.

FIG. 8D shows the sequence of Psac-D34 sequence; Pseudomonassaccharophila maltotetrahydrolase amino acid sequence with 11substitutions and deletion of the starch binding domain.

FIG. 9 shows an amino acid sequence of Pseudomonas saccharophilamaltotetrahydrolase (SEQ ID NO: 5). Pseudomonas saccharophila Glucan1,4-alpha-maltotetrahydrolase precursor (EC 3.2.1.60) (G4-amylase)(Maltotetraose-forming amylase)(Exo-maltotetraohydrolase)(Maltotetraose-forming exo-amylase).SWISS-PROT accession number P22963.

FIGS. 10A and 10B shows a nucleic acid sequence of Pseudomonassaccharophila maltotetrahydrolase (SEQ ID NO: 6). P. saccharophila mtagene encoding maltotetraohydrolase (EC number=3.2.1.60). GenBankaccession number X16732.

FIG. 11 shows an amino acid sequence of Pseudomonas stutzerimaltotetrahydrolase (SEQ ID NO:7).

FIG. 12 shows the sequence of PStu-D34 (SEQ ID NO: 8); Pseudomonasstutzeri maltotetrahydrolase amino acid sequence with substitutionsG134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N.

FIG. 13 shows the sequence of PStu-D20 (SEQ ID NO: 9); Pseudomonasstutzeri maltotetrahydrolase amino acid sequence with G134R, A141P,I157L, G223A, H307L, S334P, N33Y, D34N and G121D.

FIG. 14 shows the sequence of PStu-D14 (SEQ ID NO: 10); Pseudomonasstutzeri maltotetrahydrolase amino acid sequence with G134R, A141P,I157L, G223A, H307L, S334P, N33Y, D34N, G121D and G87S.

FIG. 15 shows the sequence of Pseudomonas stutzeri (Pseudomonasperfectomarina) (SEQ ID NO: 11). Glucan 1,4-alpha-maltotetrahydrolaseprecursor (EC 3.2.1.60) (G4-amylase) (Maltotetraose-forming amylase)(Exo-maltotetraohydrolase)(Maltotetraose-forming exo-amylase).SWISS-PROT accession number P13507.

FIG. 16 shows the sequence of Pseudomonas stutzeri maltotetrahydrolasenucleic acid sequence. P. stutzeri maltotetraose-forming amylase (amyP)gene, complete cds.

GenBank accession number M24516 (SEQ ID NO: 12).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies : A Laboratory Manual : Portable Protocol NO. I byEdward Harlow, David Lane, Ed Harlow (1999, Cold Spring HarborLaboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manualby Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring HarborLaboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson“Immunocytochemistry: Theory and Practice”, CRC Press inc., Baca Raton,Fla., 1988, ISBN 0-8493-6078-1, John D. Pound (ed); “ImmunochemicalProtocols, vol 80”, in the series: “Methods in Molecular Biology”,Humana Press, Totowa, N.J., 1998, ISBN 0-89603-493-3, Handbook of DrugScreening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes(2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref:A Handbook of Recipes, Reagents, and Other Reference Tools for Use atthe Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold SpringHarbor Laboratory, ISBN 0-87969-630-3. Each of these general texts isherein incorporated by reference.

As used herein, “PS4” shall refer to family members related to or havingsequence or functional homology with Pseudomonas saccharophilanon-maltogenic exoamylase, such as the exoamlyase having the amino acidsequence set forth in SEQ ID NO:1 or SEQ ID NO:5 or Pseudomonas stutzerinon-maltogenic exoamylase, such as a polypeptide having the amino acidsequence set forth in SEQ ID NO: 7 or SEQ ID NO11. Other family membersare set forth in Table 1.

Position numbering with respect to PS4 variants derived from Pseudomonassaccharophilia exoamylase shall be with respect SEQ ID NO: 1: 1DQAGKSPAGV RYHGGDEIIL QGFHWNVVRE APNDWYNILR QQASTIAADG FSAIWMPVPW 61RDFSSWTDGG KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDVVPNHMNR 121GYPDKEINLP AGQGFWRNDC ADPGNYPNDC DDGDRFIGGE SDLNTGHPQI YGMFRDELAN 181LRSGYGAGGF RFDFVRGYAP ERVDSWMSDS ADSSFCVGEL WKGPSEYPSW DWRNTASWQQ 241IIKDWSDRAK CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301QNGGQHHWAL QDGLIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRTAGVRAD 361SAISFHSGYS GLVATVSGSQ QTLVVALNSD LANPGQVASG SFSEAVNASN GQVRVWRSGS 421GDGGGNDGGE GGLVNVNFRC DNGVTQMGDS VYAVGNVSQL GNWSPASAVR LTDTSSYPTW 481KGSIALPDGQ NVEWKCLIRN EADATLVRQW QSGGNNQVQA AAGASTSGSFThe reference sequence is derived from the Pseudomonas saccharophiliasequence having SWISS-PROT accession number P22963, but without thesignal sequence MSHILRAAVLAAVLLPFPALA.

The numbering system, even though it may use a specific sequence as abase reference point, is also applicable to all relevant homologoussequences. For example, the position numbering may be applied tohomologous sequences from other Pseudomonas species, or homologoussequences from other bacteria. Preferably, such homologous have 60% orgreater homology, for example 70% or more, 80% or more, 90% or more or95% or more homology, with the reference sequence SEQ ID NO: 1. Sequencehomology between proteins may be ascertained using well-known alignmentprograms and hybridisation techniques described herein.

Position numbering with respect to PS4 variants derived from aPseudomonas stutzeri shall be with respect to SEQ ID NO: 7: 1 DQAGKSPNAVRYHGGDEIIL QGFHWNVVRE APNDWYNILR QQAATIAADG FSAIWMPVPW 61 RDFSSWSDGSKSGGGEGYFW HDFNKNGRYG SDAQLRQAAS ALGGAGVKVL YDVVPNHMNR 121 GYPDKEINLPAGQGFWRNDC ADPGNYPNDC DDGDRFIGGD ADLNTGHPQV YGMFRDEFTN 181 LRSQYGAGGFRFDFVRGYAP ERVNSWMTDS ADNSFCVGEL WKGPSEYPNW DWRNTASWQQ 241 IIKDWSDRAKCPVFDFALKE RMQNGSIADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHHWALQDGLIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRAAGVRAD 361 SAISFHSGYSGLVATVSGSQ QTLVVALNSD LGNPGQVASG SFSEAVNASN GQVRVWRSGT 421 GSGGGEPGALVSVSFRCDNG ATQMGDSVYA VGNVSQLGNW SPAAALRLTD TSGYPTWKGS 481 IALPAGQNEEWKCLIRNEAN ATQVRQWQGG ANNSLTPSEG ATTVGRL

As used herein, “PS4 variant nucleic acids” shall refer to nucleic acidsencoding PS4 polypeptides which are variants of PS4 family members.

As used herein, “PS4 variant polypeptides” or “PS4 variant” shall referto polypeptides which are variants of PS4 family members.

As used herein, “parent enzymes,” “parent sequence,” “parentpolypeptide” and “parent polypeptides” shall mean enzymes andpolypeptides on which the PS4 variant polypeptides are based. The parentenzyme may be a precursor enzyme (i.e. the enzyme that is actuallymutated) or it may be prepared de novo. The parent enzyme may be a wildtype enzyme.

As used herein, “variant” shall mean a molecule being derivable from aparent molecule. Variants shall include polypeptides as well as nucleicacids. Variants shall include substitutions, insertions, transversionsand inversions, among other things, at one or more locations. Variantsshall also include truncations. Variants shall include homologous andfunctional derivatives of parent molecules. Variants shall includesequences that are complementary to sequences that are capable ofhybridising to the nucleotide sequences presented herein. For example, avariant sequence is complementary to sequences capable of hybridisingunder stringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃citrate pH 7.0}) to the nucleotide sequences presentedherein. More preferably, the term variant encompasses sequences that arecomplementary to sequences that are capable of hybridising under highstringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

As used herein, “precursor” shall mean an enzyme used to produce amodified enzyme. The precursor may be an enzyme modified by mutagenesis.Likewise, the precursor may be a wild type enzyme, a variant wild typeenzyme or an already mutated enzyme.

As used herein, “functional equivalent” shall mean in relation to aparent enzyme shall mean a molecule having similar or identical functionto a parent molecule. The parent molecule may be a Pseudomonassaccharophila non-maltogenic exoamylase or a Pseudomonas stutzerinon-maltogenic exoamylase or a polypeptide obtained from other sources.The functionally equivalent enzyme may have a different amino acidsequence but will have non-maltogenic exoamylase activity. Examples ofassays to determine functionality are described herein and are known toone skilled in the art.

As used herein, “isolated” shall mean that the sequence is at leastsubstantially free from at least one other component which the sequenceis naturally associated and found in nature.

As used herein, “purified” shall mean that the sequence is in arelatively pure state—e.g. at least about 90% pure, or at least about95% pure or at least about 98% pure.

As used herein, “amylase” shall mean an enzyme that is, among otherthings, capable of catalysing the degradation of starch. Amylases arehydrolases which cleave the α-D-(1→4) O-glycosidic linkages in starch.Generally, α-amylases (E.C. 3.2.1.1, α-D-(1→4)-glucan glucanohydrolase)are defined as endo-acting enzymes cleaving α-D-(1→4) O-glycosidiclinkages within the starch molecule in a random fashion. In contrast,the exo-acting amylolytic enzymes, such as β-amylases (E.C. 3.2.1.2,α-D-(1→4)-glucan maltohydrolase) and some product-specific amylases likemaltogenic alpha-amylase (E.C. 3.2.1.133) cleave the starch moleculefrom the non-reducing end of the substrate. β-Amylases, α-glucosidases(E.C. 3.2.1.20, α-D-glucoside glucohydrolase), glucoamylase (E.C.3.2.1.3, α-D-(1→4)-glucan glucohydrolase), and product-specific amylasescan produce malto-oligosaccharides of a specific length from starch.

As used herein, “non-maltogenic exoamylase enzyme” shall mean an enzymethat does not initially degrade starch to substantial amounts ofmaltose. Assays for making such determinations are provided in herein.

As used herein, “linear malto-oligosaccharide” shall mean 2-20 units ofα-D-glucopyranose linked by an α-(1→4) bond.

As used herein, “thermostable” relates to the ability of the enzyme toretain activity after exposure to elevated temperatures. Thethermostability of an enzyme such as a non-maltogenic exoamylase ismeasured by its half-life. The half-life (t½) is the time in minutesduring which half the enzyme activity is inactivated under definedconditions. The half-life value is calculated by measuring the residualamylase activity. Half-life assays are conducted as described in moredetail in the Examples.

As used herein, “pH stable” relates to the ability of the enzyme toretain activity over a wide range of pHs. pH assays are conducted asdescribed in the Examples.

As used herein, “exo-specific” relates to an improved, e.g., increased,“exo-specificity index” as compared an exo-specificity ratio of anunsubstituted exoamylase.

As used herein, “exo-specificity index” shall mean the ratio of thetotal amylase activity to the total endoamylase activity. Assays formeasuring amylase and endoamylase activity are provided herein.

As used herein, “food” shall include both prepared food, as well as aningredient for a food, such as flour.

As used herein, “food ingredient” shall include a formulation, which isor can-be added to functional foods or foodstuffs and includesformulations used at low levels in a wide variety of products thatrequire, for example, acidifying or emulsifying. The food ingredient maybe in the form of a solution or as a solid—depending on the use and/orthe mode of application and/or the mode of administration.

As used herein, “functional food” means food capable of providing notonly -a nutritional effect and/or a taste satisfaction, but is alsocapable of delivering a further effect to consumer.

As used herein, “amino acid sequence” is synonymous with the term“polypeptide” and/or the term “protein”. In some instances, the term“amino acid sequence” is synonymous with the term “peptide”. In someinstances, the term “amino acid sequence” is synonymous with the term“enzyme”.

As used herein, “peptoid form” shall refer to variant amino acidresidues wherein the α-carbon substituent group is on the residue'snitrogen atom rather than the α-carbon. Processes for preparing peptidesin the peptoid form are known in the art, for example Simon R J et al.,PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995)13(4), 132-134.

As used herein, “nucleotide sequence” or “nucleic acid sequence” refersto an oligonucleotide sequence or polynucleotide sequence, and variant,homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand. As used herein, theterm nucleotide sequence includes genomic DNA, cDNA, synthetic DNA andRNA. Preferably it means DNA, more preferably cDNA sequence coding for aPS4 variant polypeptide.

As used herein, “starch” shall mean starch per se or a componentthereof, especially amylopectin. The term “starch medium” means anysuitable medium comprising starch. The term “starch product” means anyproduct that contains or is based on or is derived from starch.Preferably, the starch product contains or is based on or is derivedfrom starch obtained from wheat flour.

As used herein, “flour” shall mean finely-ground meal of wheat or othergrain. For example, flour may be obtained from wheat per se and not fromanother grain. Wheat flour may refer mean to wheat flour, per se, aswell as to wheat flour when present in a medium, such as dough.

As used herein, “baked farinaceous bread product ” shall mean any bakedproduct based on dough obtainable by mixing flour, water and a leaveningagent under dough forming conditions. Further components can be added tothe dough mixture.

As used herein, “homologue” and “homology” shall mean an entity having acertain degree of identity with the subject amino acid sequences and thesubject nucleotide sequences. A homologous sequence is taken to includean amino acid sequence at least 75, 80, 85 or 90% identical, preferablyat least 95, 96, 97, 98 or 99% identical to the subject sequence.Typically, homologues will comprise the same active sites as the subjectamino acid sequence.

As used herein, “hybridisation” shall include the process by which astrand of nucleic acid joins with a complementary strand through basepairing as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies. The PS4 nucleic acid mayexist as single- or double-stranded DNA or RNA, an RNA/DNA heteroduplexor an RNA/DNA copolymer.

As used herein, “copolymer” refers to a single nucleic acid strand thatcomprises both ribonucleotides and deoxyribonucleotides. The PS4 nucleicacid may even be codon optimised to further increase expression.

As used herein, “synthetic” shall refer to that which is produced by invitro chemical or enzymatic synthesis. It includes but is not limited toPS4 nucleic acids made with optimal codon usage for host organisms suchas the methylotrophic yeasts Pichia and Hansenula

As used herein, “transformed cell” shall include cells that have beentransformed by use of recombinant DNA techniques. Transformationtypically occurs by insertion of one or more nucleotide sequences into acell. The inserted nucleotide sequence may be a heterologous nucleotidesequence (i.e. is a sequence that is not natural to the cell that is tobe transformed. In addition, or in the alternative, the insertednucleotide sequence may be an homologous nucleotide sequence, i.e. is asequence that is natural to the cell that is to be transformed)—so thatthe cell receives one or more extra copies of a nucleotide sequencealready present in it.

As used herein, “operably linked” shall mean that the componentsdescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence operably linked to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under condition compatible with the control sequences.

As used herein, “biologically active” shall refer to a sequence having asimilar structural function (but not necessarily to the same degree),and/or similar regulatory function (but not necessarily to the samedegree) and/or similar biochemical function (but not necessarily to thesame degree) of the naturally occurring sequence.

I. Detailed Description of the Polypeptides of the Invention

In a first aspect, the invention provides a polypeptide comprising a PS4variant, the PS4 variant being derivable from a parent polypeptide. Theparent enzyme may preferably be a non-maltogenic exoamylase, preferablybacterial non-maltogenic exoamylase enzyme. The parent enzyme maypreferably be a polypeptide which exhibits non-maltogenic exoamylaseactivity.

The parent enzyme may be a Pseudomonas saccharophila non-maltogenicexoamylase, such as the exoamlyase set forth in SEQ ID NO:1 or SEQ IDNO:5. The parent enzyme may be a Pseudomonas stutzeri non-maltogenicexoamylase, such as a polypeptide having set forth in SEQ ID NO: 7 orSEQ ID NO11. Other members of the P54 family may be used as parentenzymes as set forth in Table 1 below. Preferably, P54 family memberswill generally be similar to, homologous to or functionally equivalentto the exoamlyases set forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7 orSEQ ID NO:11, and may be identified by standard methods, such ashybridisation screening of a suitable library using probes, or by genomesequence analysis. Methods of identification are set forth below. TABLE1 Parent Sequences (PS4 family members). The sequences depicted differfrom the Pseudomonas saccharophila sequence at the positions shown onthe top row of the table, by including substitutions consisting of theamino acid residues set out. For example, pS4cc1-S161A is a variant ofwild type Pseudomonas non-maltogenic exo-amylase, and thus can be usedas a parent enzyme. Furthermore, non-maltogenic exoamylases from otherstrains of Pseudomonas spp, such as ATCC17686, may also be used as aparent polypeptide. The PS4 variant polypeptide residues may be insertedinto any of these parent sequences to generate the variant PS4polypeptide sequences

The PS4 variant polypeptide varies from the parent sequence by includinga number of mutations comprising amino acid substitutions. In preferredembodiment, the parent polypeptide is a non-maltogenic exoamylase fromPseudomonas saccharophilia non-maltogenic exoamylase having a sequenceset forth in SEQ ID NO: 1 or set forth in SEQ ID NO:5. In otherpreferred embodiments, the parent polypeptide comprises a non-maltogenicexoamylase from Pseudomonas stutzeri having a sequence shown set forthin SEQ ID NO:7 or set forth in SEQ ID NO:11. In a preferred embodiment,the PS4 variant differs from the parent polypeptide by including aminoacid substitutions, the substitutions located at a position comprisingat least one position selected from the group consisting of: 4, 9, 13,33, 34, 42, 70, 71, 87, 99, 100, 108, 113, 121, 131, 134, 135, 141, 153,157, 158, 160, 161, 166, 170, 171, 178, 179, 184, 188, 198, 199, 221,223, 238, 270, 277, 290, 307, 315, 334, 335, 342, 343, 372, 392, 398,399, 405, 415, 425, wherein reference to position numbering is withrespect to a Pseudomonas saccharophilia exoamylase sequence shown as SEQID NO: 1. Preferably, the position is at least one position selectedfrom the group consisting of: 33, 34, 87, 121, 134, 141, 157, 178, 179,223, 307 and 334. In another embodiment the variant further comprises asubstitution selected from the group of 71, 108, 158, 171, 188, and 343.In another embodiment the variant further comprises a substitutionselected from the group of 113, 198, and 290.

Preferably, the PS4 variant comprises at least one substitution selectedfrom the group consisting of: N33Y, D34N, G87S, G121D, G134R, A141P,I157L, L178F, A179T, G223A, H307L and S334P. Preferably, the PS4 variantcomprises the following substitutions: N33Y, D34N, G134R, A141P, I157L,G223A, H307L and S334P with at least one additional substitution ofL178F or A179T. Preferably, the PS4 variant comprises one of thefollowing substitutions: N33Y, D34N, I157L, L178F, A179T, G223A orH307L. Preferably, the PS4 variant comprises one of the followingsubstitutions: G87S, G134R, A141P, or S334P. In another embodiment, thePS4 variant comprises one of the following substitutions: K71R, K108R,G158D, Y171S, G188A, and D343E. In another embodiment, the PS4 variantcomprises one of the following substitutions: V113I, Y198F, Y198W, andV290I.

While not wanting to be bound by theory, it is proposed by the inventorsthat three-dimensional crystal structure of Pseudomonas amylase inconjunction with a proposed substrate model binding site (indicated byMolecular Operating Environment [MOE} software program available fromChemical Computing Group, Inc., Montreal Canada) indicates residuepositions 121, 157, 223, and 307 would be in close proximity to thesubstrate binding site. Since data as shown in the examples demonstratesthat G121D improves both the stability of the enzyme and theexo-specificity and G223A improves the enzyme thermostability, given theimprovements already observed, and the positions expected proximity tothe substrate binding site, further improvement can be obtained bymaking all possible amino acid replacements at each such position. Inone embodiment, close proximity refers to the particular positions beingwithin 10.0 angstroms of the substrate binding site. In anotherembodiment, close proximity refers to the particular positions beingwithin 7.5 angstroms of the substrate binding site. In one embodiment,close proximity refers to the particular positions being within 6.0angstroms of the substrate binding site, e.g., G121 C-alpha to thesubstrate about 5.9 anstroms; G223 C-alpha to substrate about 5.82angstroms.

In one embodiment, the PS4 variant comprises a combination selected fromthe following groups of:

-   G134R, A141P, I157L, G223A, H307L, S334P, D343E, and G121D;-   G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, and G121D;-   G134R, A141P, I157L, G223A, H307L, S334P, D343E, and N33Y;-   G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R,    L178F, A179T, G87S, G121D, S214N, and T375A;-   G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R,    L178F, A179T, G121D, Y171S, G188A, and N138D;-   G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R,    L178F, A179T, and G121D;-   G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y,    D34N, K71R, L178F, and A179T;-   G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y,    D34N, K71R, L178F, A179T, and G188A;-   G134R, A141P, I157L, G223A, H307L, S334P, K71R, L178F, and A179T;-   G134R, A141P, I157L, G223A, H307L, S334P, L178F, and A179T;-   G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F, and    A179T;-   G134R, A141P, I157L, G223A, H307L, S334P, L178F, A179T, G87S, and    G121D;-   G134R, A141P, I157L, G223A, H307L, S334P, L178F, A179T, and G121D;-   G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, K71R, L178F,    A179T, and G121D;-   G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R    L178F A179T;-   G87S G121D G134R, A141P I157L G223A H307L S334P K71R L178F A179T;-   G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F    A179T G121D; and-   G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D.

In another embodiment, the at least one substitution comprises acombination selected from the group of:

-   A141P, I157L, G223A, H307L and S334P;-   G121D, G134R, A141P, I157L, G223A, H307L and S334;-   G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P;-   G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;-   G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;-   G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;-   N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;-   N33Y, D34N, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L    and S334P; and-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P.

In another embodiment, the exoamylase comprising at least onesubstitution comprises a combination selected from the following:

-   N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and    S334P;-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P; and-   N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A,    H307L and S334P.

In another embodiment, the exoamylase comprising at least onesubstitution comprises a combination selected from the following:

-   G87S, V113I G134R, A141P, I157L, Y198F, G223A, V290I H307L, S334P,    and D343E;-   113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E;-   A99V, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and    D343E; V113I, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and    D343E;-   A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E;-   A199V, D343E, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, and    S334P;-   V113I, A141P, I157L, Y198F, G223A, V290I, S334P, and D343E;

V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,andD343E;

-   V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P, and D343E;-   V113I, A141 P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E;-   V113I, A141P, Y198F, G223A, V290I, and H307L;-   V131I, A141P, Y 198F, G223A, V290I, S334P, and D343E; V131I, A141 P,    Y198F, G223A, A268P, V290I, and S399P-   V113I, A141 P, Y198F, G223A, V290I, and S399P;-   V113I, A141P, Y198W, G223A, and V290I;-   V113I, A141P, Y198F, G223A, and V290I;-   Y198F, G223A, and V290I;-   Y198W, G223A, and V290I;-   V113I, A141P, I157L, Y198F, G223A, and V290I;-   V113M;-   V113A;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and    D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, I315V,    S334P, and D343E;

D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, andD343E;

-   V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L,    S334P, and D343E;-   K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L,    G313G, S334P, and D343E;-   D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G,    S334P, and D343E;-   V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L,    S334P, and D343

V113I, G134R, A141P, I157L,11701, Y198F, G223A, V290I, H307L, G313G;S334P, and D343E

-   V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L,    G313G, S334P, and D343E;-   G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    and D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E, and A405E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    D343E, and A405V;-   A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E;-   V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P, and    D343E;-   V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and    D343E;

0 K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,and D343E;

-   K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,    S334P, and D343E;-   D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,    and D343E;-   G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and    D343E;-   A141 P, G1 34R, G223A, H307L, I157L, V113I, V290I, Y198F, and G188A;    In preferred embodiments, the PS4 variant is an amino acid sequence    comprising either SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO    4a, SEQ ID NO 4b, SEQ ID NO 4c, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID    NO:10.

The PS4 variant polypeptide may comprise one or more mutations inaddition to those set out above. Other mutations, such as deletions,insertions, substitutions, transversions, transitions and inversions, atone or more other locations, may also be included. Likewise, thepolypeptide may be missing at least one of the substitutions set forthabove.

The PS4 variant may also comprise a conservative substitution that mayoccur as a like-for-like substitution (e.g., basic for basic, acidic foracidic, polar for polar etc.) Non-conservative substitution may alsooccur i.e. from one class of residue to another or alternativelyinvolving the inclusion of unnatural amino acids such as ornithine(hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in amino acid properties (such aspolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues) and it is therefore useful to groupamino acids together in functional groups. Amino acids can be groupedtogether based on the properties of their side chain alone. However itis more useful to include mutation data as well. The sets of amino acidsthus derived are likely to be conserved for structural reasons. Thesesets can be described in the form of a Venn diagram (Livingstone C. D.and Barton G. J. (1993) “Protein sequence alignments: a strategy for thehierarchical analysis of residue conservation” Comput.Appl Biosci. 9:745-756)(Taylor W. R. (1986) “The classification of amino acidconservation” J. Theor.Biol. 119; 205-218). Conservative substitutionsmay be made, for example according to the table below which describes agenerally accepted Venn diagram grouping of amino acids. Set Sub-setHydrophobic F W Y H K M I L V Aromatic F W Y H A G C Aliphatic I L VPolar W Y H K R E D C S Charged H K R E D T N Q Positively H K R chargedNegatively E D charged Small V C A G S P T N D Tiny A G S

Variant amino acid sequences may also include suitable spacer groupsinserted between any two amino acid residues of the sequence includingalkyl groups such as methyl, ethyl or propyl groups in addition to aminoacid spacers such as glycine or β-alanine residues. A further form ofvariation involves the presence of one or more amino acid residues inpeptoid form.

The PS4 variant may also comprise a homologous sequence. A homologoussequence comprises a nucleotide sequence at least 75, 80, 85 or 90%identical, preferably at least 95, 96, 97, 98 or 99% identical to anucleotide sequence encoding a PS4 variant. Typically, the homologueswill comprise the same sequences that code for the active sites as thesubject sequence.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences. % homology may be calculated overcontiguous sequences, i.e. one sequence is aligned with the othersequence and each amino acid in one sequence is directly compared withthe corresponding amino acid in the other sequence one residue at atime. This is called an “ungapped” alignment. Typically, such ungappedalignments are performed only over a relatively short number ofresidues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so,that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap., This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer-program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc.Acids Research 12 p387). Examples of other software than can performsequence comparisons include, but are not limited to, the BLAST package(see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4^(th)Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) andthe GENEWORKS suite of comparison tools. Both BLAST and FASTA areavailable for offline and online searching (see Ausubel et al., 1999,Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, forsome applications, it is preferred to use the GCG Bestfit program. BLAST2 Sequences is also available for comparing protein and nucleotidesequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS MicrobiolLett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe s case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in DNASIS™ (Hitachi Software), based on analgorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Preferred embodiments also include functional equivalents. The PS4variant polypeptides described in this document are derived from, or arevariants of, polypeptides which preferably exhibit non-maltogenicexoamylase activity. Preferably,.these parent enzymes are non-maltogenicexoamylases themselves. The PS4 variant polypeptides themselves inpreferred embodiments also exhibit non-maltogenic exoamylase activity.

The PS4 variants described here will preferably have exospecificity, forexample measured by exo-specificity indices, as described above,consistent with their being exoamylases. Moreover, they preferably havehigher or increased exospecificity when compared to the parent enzymesor polypeptides from which they are derived. Thus, for example, the PS4variant polypeptides may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200% or higher exo-specificity index when compared to theirparent polypeptides, preferably under identical conditions. They mayhave 1.5× or higher, 2× or higher, 5× or higher, 10× or higher, 50× orhigher, 100× or higher, when compared to their parent polypeptides,preferably under identical conditions.

In preferred embodiments, the functional equivalents will have sequencehomology to at least one of the PS4 family members. Functionalequivalents will have sequence homology to either of the Pseudomonassaccharophila and Pseudomonas stutzeri non-maltogenic exoamylasesmentioned above, preferably both. The functional equivalent may alsohave sequence homology with any of the sequences set out as SEQ ID NOs:1 to 12, preferably SEQ ID NO: 1 or SEQ ID NO: 7 or both. Sequencehomology between such sequences is preferably at least 60%, preferably65% or more, preferably 75% or more, s preferably 80% or more,preferably 85% or more, preferably 90% or more, preferably 95% or more.

In other embodiments, the functional equivalents will be capable ofspecifically hybridising to any of the sequences set out above. Methodsof determining whether one sequence is capable of hybridising to anotherare known in the art, and are for example, described in Sambrook et al(supra) and Ausubel, F. M. et al. (supra). In highly preferredembodiments, the functional equivalents will be capable of hybridisingunder stringent conditions, e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0}.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques. Several methods are described below.

2. Detailed Description of the Nucleic Acids of the Invention

In a second aspect, the invention provides a nucleic acid, the nucleicacid encoding a polypeptide comprising a PS4 variant being derivablefrom a parent polypeptide, as set forth above.

One skilled in the art will be aware of the relationship between nucleicacid sequence and polypeptide sequence, in particular, the genetic codeand the degeneracy of this code, and will be able to construct such PS4nucleic acids without difficulty. For example, one skilled in the artwill be aware that for each amino acid substitution in the PS4 variantpolypeptide sequence there may be one or more codons which encode thesubstitute amino acid. Accordingly, it will be evident that, dependingon the degeneracy of the genetic code with respect to that particularamino acid residue, one or more PS4 nucleic acid sequences may begenerated corresponding to that PS4variant polypeptide sequence. Thus,for example, a PS4 variant nucleic acid sequence may be derivable from aparent sequence encoding a polypeptide having, wherein the PS4 variantnucleic acid encodes amino acid substitutions at the followingpositions: G134, A141, I157, G223, H307, S334, N33 and D34, togetherwith one or both of L178 and A179.

Mutations in amino acid sequence and nucleic acid sequence may be madeby any of a number of techniques, as known in the art. In particularlypreferred embodiments, the mutations are introduced into parentsequences by means of PCR (polymerase chain reaction) using appropriateprimers, as illustrated in the Examples.

The parent enzymes may be modified at the amino acid level or thenucleic acid level to generate the PS4 variant sequences describedherein. Therefore, a preferred embodiment of this aspect of theinvention provides for the generation of PS4 variant polypeptides byintroducing one or more corresponding codon changes in the nucleotidesequence encoding a non-maltogenic exoamylase polypeptide.

It will be appreciated that the above codon changes can be made in anyPS4 family nucleic acid sequence. For example, sequence changes can bemade to a Pseudomonas saccharophila or a Pseudomonas stutzerinon-maltogenic exoamylase nucleic acid sequence (e.g., X16732, SEQ IDNO: 6 or M24516, SEQ ID NO: 12).

The parent enzyme may comprise the “complete” enzyme, i.e., in itsentire length as it occurs in nature (or as mutated), or it may comprisea truncated form thereof. The PS4 variant derived from such mayaccordingly be so truncated, or be “full-length”. The truncation may beat the N-terminal end or the C-terminal end. The parent enzyme or PS4variant may lack one or more portions, such as sub-sequences, signalsequences, domains or moieties, whether active or not. For example, theparent enzyme or the-PS4 variant polypeptide may lack a signal sequence,as described above. Alternatively, or in addition, the parent enzyme orthe PS4 variant may lack one or more catalytic or binding domains.

In highly preferred embodiments, the parent enzyme or PS4 variant maylack one or more of the domains present in non-maltogenic exoamylases,such as the starch binding domain. For example, the PS4 polypeptides mayhave only sequence up to position 429, relative to the numbering of aPseudomonas saccharophilia non-maltogenic exoamylase shown as SEQ IDNO: 1. For example, this is the case for the PS4 variants pSac-d34,pSac-D20, pSac-D14 or the other variants described in the Examples.

Typically, the PS4 variant nucleotide sequence is prepared usingrecombinant DNA techniques. However, in an alternative embodiment, thenucleotide sequence could be synthesised, in whole or in part, usingchemical methods well known in the art (see Caruthers M H et al., (1980)Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc. Acids ResSymp Ser 225-232).

A nucleotide sequence encoding either an enzyme which has the specificproperties as defined herein or an enzyme which is suitable formodification, such as a parent enzyme, may be identified and/or isolatedand/or purified from any cell or organism producing said enzyme. Variousmethods are well known within the art for the identification and/orisolation and/or purification of nucleotide sequences. By way ofexample, PCR amplification techniques to prepare more of a sequence maybe used once a suitable sequence has been identified and/or isolatedand/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the enzyme. If the amino acid sequence of the enzyme or a partof the amino acid sequence of the enzyme is known, labelledoligonucleotide probes may be synthesised and used to identifyenzyme-encoding clones from the genomic library prepared from theorganism. Alternatively, a labelled oligonucleotide probe containingsequences homologous to another known enzyme gene could be used toidentify enzyme-encoding clones. In the latter case, hybridisation andwashing conditions of lower stringency are used.

Alternatively, enzyme-encoding clones could be identified by insertingfragments of genomic DNA into an expression vector, such as a plasmid,transforming enzyme-negative bacteria with the resulting genomic DNAlibrary and then plating the transformed bacteria onto agar platescontaining a substrate for enzyme (e.g., maltose), thereby allowingclones expressing the enzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding theenzyme may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869 or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin inaccordance with standard techniques. Each ligated fragment correspondsto various parts of the entire nucleotide sequence. The DNA sequence mayalso be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or inSaiki R K et al., (Science (1988) 239, pp 487-491).

The nucleotide sequences described here, and suitable for use in themethods and compositions described here may include within themsynthetic or modified nucleotides. A number of different types ofmodification to oligonucleotides are known in the art. These includemethylphosphonate and phosphorothioate backbones and/or the addition ofacridine or polylysine chains at the 3′ and/or 5′ ends of the molecule.For the purposes of this document, it is to be understood that thenucleotide sequences described herein may be modified by any methodavailable in the art. Such modifications may be carried out in order toenhance the in vivo activity or life span of nucleotide sequences.

A preferred embodiment of the invention provides for nucleotidesequences and the use of nucleotide sequences that are complementary tothe sequences presented herein, or any derivative, fragment orderivative thereof. If the sequence is complementary to a fragmentthereof then that sequence can be used as a probe to identify similarcoding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the PS4 variantsequences may be obtained in a number of ways. Other variants of thesequences described herein may be obtained for example by probing DNAlibraries made from a range of individuals, for example individuals fromdifferent populations. In addition, other homologues maybe obtained andsuch homologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other species and probing suchlibraries with probes comprising all or part of any one of the sequencesin the attached sequence listings under conditions of medium to highstringency. Similar considerations apply to obtaining species homologuesand allelic variants of the polypeptide or nucleotide sequencesdescribed here.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences. The primers used in degenerate PCR will contain one or moredegenerate positions and will be used at stringency conditions lowerthan those used for cloning sequences with single sequence primersagainst known sequences. Conserved sequences can be predicted, forexample, by aligning the amino acid sequences from severalvariants/homologues. Sequence alignments can be performed using computersoftware known in the art as described herein.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences. This may be useful where, forexample, silent codon sequence changes are required to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

The polynucleotides may be used to produce a primer, e.g. a PCR primer,a primer for an alternative amplification reaction, a probe e.g.labelled with a revealing label by conventional means using radioactiveor non-radioactive labels or the polynucleotides may be cloned intovectors. Such primers, probes and other fragments will be at least 15,preferably at least 20, for example at least 25, 30 or 40 nucleotides inlength, and are also encompassed by the term polynucleotides.

Polynucleotides such as DNA polynucleotides and probes may be producedrecombinantly, synthetically or by any means available to those of skillin the art. They may also be cloned by standard techniques. In general,primers will be produced by synthetic means, involving a stepwisemanufacture of the desired nucleic acid sequence one nucleotide at atime. Techniques for accomplishing this using automated techniques arereadily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. The primers may be designed to contain suitable restrictionenzyme recognition sites so that the amplified DNA can be cloned into asuitable cloning vector. Preferably, the variant sequences are at leastas biologically active as the sequences presented herein.

A preferred embodiment of the invention includes sequences that arecomplementary to the nucleic acid sequences of PS4 variants or sequencesthat are capable of hybridising either to the nucleotide sequences ofPS4 variants (including complementary sequences of those presentedherein), as well as nucleotide sequences that are complementary tosequences that can hybridise to the nucleotide sequences of PS4 variants(including complementary sequences of those presented herein). Apreferred embodiment provides polynucleotide sequences that are capableof hybridising to the nucleotide sequences presented herein underconditions of intermediate to maximal stringency.

A preferred embodiment includes nucleotide sequences that can hybridiseto the nucleotide sequence of a PS4 variant nucleic acid, or thecomplement thereof, under stringent conditions (e.g. 50° C. and0.2×SSC). More preferably, the nucleotide sequences can hybridise to thenucleotide sequence of a PS4 variant, or the complement thereof, underhigh stringent conditions (e.g. 65° C. and 0.1×SSC).

Once an enzyme-encoding nucleotide sequence has been isolated, or aputative enzyme-encoding nucleotide sequence has been identified, it maybe desirable to mutate the sequence in order to prepare an enzyme.Accordingly, a PS4 variant sequence may be prepared from a parentsequence. Mutations may be introduced using synthetic oligonucleotides.These oligonucleotides contain nucleotide sequences flanking the desiredmutation sites. A suitable method is disclosed in Morinaga et al.,(Biotechnology (1984) 2, p 646-649). Another method of introducingmutations into enzyme-encoding nucleotide sequences is described inNelson and Long (Analytical Biochemistry (1989), 180, p 147-151). Afurther method is described in Sarkar and Sommer (Biotechniques (1990),8, p 404-407—“The megaprimer method of site directed mutagenesis”).

In a preferred embodiment, the sequence for use in the methods andcompositions described here is a recombinant sequence—i.e. a sequencethat has been prepared using recombinant DNA techniques. Such techniquesare explained, for example, in the literature, for example, J. Sambrook,E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.

3. Detailed Description of Compositions of the Invention

A third aspect of the invention provides for compositions comprisingpolypeptides which are variants of polypeptides having non-maltogenicexoamylase activity, as well as uses of such variant polypeptides andthe compositions. The compositions include the polypeptide variantstogether with another component.

A preferred embodiment of the invention comprises a PS4 variantpolypeptide, optionally together with a further ingredient or a furtherenzyme or both. In addition to the PS4 variant polypeptides, one or moreenzymes may be added, for example added to the food, dough preparation,foodstuff or starch composition. Further enzymes that may be added tothe dough include oxidoreductases, hydrolases, such as lipases (e.g.,lipase (EC 3.1.1) capable of hydrolysing carboxylic ester bonds torelease carboxylate or such as triacylglycerol lipase (EC 3.1.1.3),galactolipase (EC 3.1.1.26), phospholipase A1 (EC 3.1.1.32),phospholipase A2 (EC 3.1.1.4) and lipoprotein lipase A2 (EC 3.1.1.34))and esterases as well as glycosidases like α-amylase, pullulanase andxylanase. Oxidoreductases, such as maltose oxidising enzyme, a glucoseoxidase (EC 1.1.3.4), carbohydrate oxidase, glycerol oxidase, pyranoseoxidase, galactose oxidase (EC 1.1.3.10) and hexose oxidase (EC 1.1.3.5) can be used for dough strengthening and control of volume of thebaked products and, xylanases and other hemicellulases may be added toimprove dough handling properties, crumb softness and bread volume.Lipases are useful as dough strengtheners and crumb softeners andac-amylases and other amylolytic enzymes may be incorporated into thedough to control bread volume and further reduce crumb firmness. Furtherenzymes that may be used can be selected from the group consisting of acellulase, a hemicellulase, a starch degrading enzyme, a protease, alipoxygenase. In a preferred embodiment, a PS4 variant polypeptide maybe combined with amylases, in particular, maltogenic amylases.Maltogenic alpha-amylase (glucan 1,4-a-maltohydrolase, E.C. 3.2.1.133)is able to hydrolyze amylose and amylopectin to maltose in thealpha-configuration.

A maltogenic alpha-amylase from Bacillus (EP 120 693) is commerciallyavailable ( Novo Nordisk A/S, Denmark) and is widely used in the bakingindustry-as an anti-staling agent due to its ability to reduceretrogradation of starch. (see, for example, WO 91/04669). Themaltogenic alpha-amylase shares several characteristics withcyclodextrin glucanotransferases (CGTases), including sequence homology(Henrissat B, Bairoch A; Biochem. J., 316, 695-696 (1996)) and formationof transglycosylation products (Christophersen, C., et al., 1997,Starch, vol. 50, No. 1, 39-45). A preferred embodiment includescombinations comprising PS4 variant polypeptides together with thealpha-amylase or any of its variants. Such combinations are useful forfood production such as baking. Variants, homologues, and mutants ofvariants disclosed in U.S. Pat. No. 6,162,628, the entire disclosure ofwhich is hereby incorporated by reference, may be used in combinationwith the PS4 variant polypeptides described herein. In particular, anyof the polypeptides described in that document, specifically variants ofSEQ ID NO: 1 of U.S. Pat. No. 6,162,628 at any one or more positionscorresponding to Q13, I16, D17, N26, N28, P29, A30, S32, Y33, G34, L35,K40, M45, P73, V74, D76 N77, D79, N86, R95, N99, I100, H103, Q119, N120,N131, S141, T142, A148, N152, A163, H169, N171, G172, I174, N176, N187,F188, A192, Q201, N203, H220, N234, G236, Q247, K249, D261, N266, L268,R272, N275, N276, V279, N280, V281, D285, N287, F297, Q299, N305, K316,N320, L321, N327, A341, N342, A348, Q365, N371, N375, M378, G397, A381,F389, N401, A403, K425, N436, S442, N454, N468, N474, S479, A483, A486,V487, S493, T494, S495, A496, S497, A498, Q500, N507, 1510, N513, K520,Q526, A555, A564, S573, N575, Q581, S583, F586, K589, N595, G618, N621,Q624, A629, F636, K645, N664, and/or T681 may be used.

The further enzyme can be added together with any dough ingredientincluding the flour, water or optional other ingredients or additives orthe dough improving composition. The further enzyme can be added beforeor after the flour, water and optionally other ingredients and additivesor the dough improving composition. The further enzyme may convenientlybe a liquid preparation or in the form of a dry composition.

Some enzymes of the dough improving composition are capable ofinteracting with each other under the dough conditions to an extentwhere the effect on improvement of the rheological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromdough by the enzymes is not only additive, but the effect issynergistic. In relation to improvement of the product made from dough(finished product), it may be found that the combination results in asubstantial synergistic effect in respect to crumb structure. Also, withrespect to the specific volume of baked product a synergistic effect maybe found.

4. Vectors, Cells and Methods of Expressing a PS4 Polypeptide

A fourth aspect provides vectors comprising a PS4 variant polypeptide,cells comprising a PS4 variant polypeptide and methods of expressing aPS4 variant polypeptide.

The nucleotide sequence for use in the methods and compositionsdescribed herein may be incorporated into a recombinant replicablevector. The vector may be used to replicate and express the nucleotidesequence, in enzyme form, in and/or from a compatible host cell.Expression may be controlled using control sequences, eg. regulatorysequences. The enzyme produced by a host recombinant cell by expressionof the nucleotide sequence may be secreted or may be containedintracellularly depending on the sequence and/or the vector used. Thecoding sequences may be designed with signal sequences which directsecretion of the substance coding sequences through a particularprokaryotic or eukaryotic cell membrane.

Polynucleotides can be incorporated into a recombinant replicablevector. The vector may be used to replicate the nucleic acid in acompatible host cell. The vector comprising the polynucleotide sequencemay be transformed into a suitable host cell. Suitable hosts may includebacterial, yeast, insect and fungal cells.

PS4 variant polypeptides and polynucleotides may be expressed byintroducing a polynucleotide into a replicable vector, introducing thevector into a compatible host cell and growing the host cell underconditions which bring about replication of the vector. The vector maybe recovered from the host cell.

The PS4 nucleic acid may be operatively linked to transcriptional andtranslational regulatory elements active in a host cell of interest. ThePS4 nucleic acid may also encode a fusion protein comprising signalsequences such as, for example, those derived from the glucoamylase genefrom Schwanniomyces occidentalis, α-factor mating type gene fromSaccharomyces cerevisiae and the TAKA-amylase from Aspergillus oryzae.Alternatively, the PS4 nucleic acid may encode a fusion proteincomprising a membrane binding domain.

The PS4 variant may be expressed at the desired levels in a hostorganism using an expression vector. An expression vector comprising aPS4 nucleic acid can be any vector is capable of expressing the geneencoding the PS4 nucleic acid in the selected host organism, and thechoice of vector will depend on the host cell into which it is to beintroduced. Thus, the vector can be an autonomously replicating vector,i.e. a vector that exists as an episomal entity, the replication ofwhich is independent of chromosomal replication, such as, for example, aplasmid, a bacteriophage or an episomal element, a minichromosome or anartificial chromosome. Alternatively, the vector may be one which, whenintroduced into a host cell, is integrated into the host cell genome andreplicated together with the chromosome.

The expression vector typically includes the components of a cloningvector, such as, for example, an element that permits autonomousreplication of the vector in the selected host organism and one or morephenotypically detectable markers for selection purposes. The expressionvector normally comprises control nucleotide sequences encoding apromoter, operator, ribosome binding site, translation initiation signaland optionally, a repressor gene or one or more activator genes.Additionally, the expression vector may comprise a sequence coding foran amino acid sequence capable of targeting the PS4 variant to a hostcell organelle such as a peroxisome or to a particular host cellcompartment. Such a targeting sequence includes but is not limited tothe sequence SKL. For expression under the direction of controlsequences, the nucleic acid sequence the PS4 variant is operably linkedto the control sequences in proper manner with respect to expression.

Preferably, a polynucleotide in a vector is operably linked to a controlsequence that is capable of providing for the expression of the codingsequence by the host cell, i.e. the vector is an expression vector. Thecontrol sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators. The control sequences may in particularcomprise promoters.

In the vector, the nucleic acid sequence encoding for the variant PS4polypeptide is operably combined with a suitable promoter sequence. Thepromoter can be any DNA sequence having transcription activity in thehost organism of choice and can be derived from genes that arehomologous or heterologous to the host organism. Examples of suitablepromoters for directing the transcription of the modified nucleotidesequence, such as PS4 nucleic acids, in a bacterial host include thepromoter of the lac operon of E. coli, the Streptomyces coelicoloragarase gene dagA promoters, the promoters of the Bacillus licheniformisα-amylase gene (amyL), the aprE promoter of Bacillus subtilis, thepromoters of the Bacillus stearothermophilus maltogenic amylase gene(amyM), the promoters of the Bacillus amyloliquefaciens α-amylase gene(amyQ), the promoters of the Bacillus subtilis xylA and xylB genes and apromoter derived from a Lactococcus sp.-derived promoter including theP170 promoter. When the gene encoding the PS4 variant polypeptide isexpressed in a bacterial species such as E. coli, a suitable promotercan be selected, for example, from a bacteriophage promoter including aT7 promoter and a phage lambda promoter. For transcription in a fungalspecies, examples of useful promoters are those derived from the genesencoding the, Aspergillus oryzae TAKA amylase, Rhizomucor mieheiaspartic proteinase, Aspergillus niger neutral α-amylase, A. niger acidstable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase or Aspergillus nidulans acetamidase. Examples ofsuitable promoters for the expression in a yeast species include but arenot limited to the Gal 1 and Gal 10 promoters of Saccharomycescerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.

Examples of suitable bacterial host organisms are gram positivebacterial species such as Bacillaceae including Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillusthuringiensis, Streptomyces species such as Streptomyces murinus, lacticacid bacterial species including Lactococcus spp. such as Lactococcuslactis, Lactobacillus spp. including Lactobacillus reuteri, Leuconostocspp., Pediococcus spp. and Streptococcus spp. Alternatively, strains ofa gram-negative bacterial species belonging to Enterobacteriaceaeincluding E. coli, or to Pseudomonadaceae can be selected as the hostorganism. A suitable yeast host organism can be selected from thebiotechnologically relevant yeasts species such as but not limited toyeast species such as Pichia sp., Hansenula sp or Kluyveromyces,Yarrowinia species or a species of Saccharomyces including Saccharomycescerevisiae or a is species belonging to Schizosaccharomyce such as, forexample, S. Pombe species. Preferably a strain of the methylotrophicyeast species Pichia pastoris is used as the host organism. Preferablythe host organism is a Hansenula species. Suitable host organisms amongfilamentous fungi include species of Aspergillus, e.g. Aspergillusniger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamorior Aspergillus nidulans. Alternatively, strains of a Fusarium species,e.g. Fusarium oxysporum or of a Rhizomucor species such as Rhizomucormiehei can be used as the host organism. Other suitable strains includeThermomyces and Mucor species.

Host cells comprising polynucleotides may be used to expresspolypeptides, such as variant PS4 polypeptides, fragments, homologues,variants or derivatives thereof. Host cells may be cultured undersuitable conditions which allow expression of the proteins. Expressionof the polypeptides may be constitutive such that they are continuallyproduced, or inducible, requiring a stimulus to initiate expression. Inthe case of inducible expression, protein production can be initiatedwhen required by, for example, addition of an inducer substance to theculture medium, for example dexamethasone or IPTG. Polypeptides can beextracted from host cells by a variety of techniques known in the art,including enzymatic, chemical and/or osmotic lysis and physicaldisruption. Polypeptides may also be produced recombinantly in an invitro cell-free system, such as the TnT™ (Promega) rabbit reticulocytesystem.

5. Use(s)

In the following description and examples, unless the context dictatesotherwise, dosages of PS4 variant polypeptides are given in parts permillion (micrograms per gram) of flour. For example, “1 D34” as used inTable 2 indicates 1 part per million of pSac-D34.

The PS4 substitution mutants described here may be used for any purposefor which the parent enzyme is suitable. In particular, they may be usedin any application for which exo-maltotetraohydrolase is used. In highlypreferred embodiments, they have the added advantage of higherthermostability, or higher exoamylase activity or higher pH stability,or any combination. Examples of suitable uses for the PS4 variantpolypeptides and nucleic acids include food production, in particularbaking, as well as production of foodstuffs; further examples are setout in detail below.

The following system is used to characterize polypeptides havingnon-maltogenic exoamnylase activity which are suitable for use accordingto the methods and compositions described here. This system may forexample be used to characterise the PS4 parent or variant polypeptidesdescribed here.

By way of initial background information, waxy maize amylopectin(obtainable as WAXILYS 200 from Roquette, France) is a starch with avery high amylopectin content (above 90%). 20 mg/ml of waxy maize starchis boiled for 3 min. in a buffer of 50 mM MES(2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 andsubsequently incubated at 50° C. and used within half an hour.

One unit of the non-maltogenic exoamylase is defined as the amount ofenzyme which releases hydrolysis products equivalent to 1 μmol ofreducing sugar per min. when incubated at 50 degrees C. in a test tubewith 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calciumchloride, pH 6.0 prepared as described above. Reducing sugars aremeasured using maltose as standard and using the dinitrosalicylic acidmethod of Bernfeld, Methods Enzymol., (1954), 1, 149-158 or anothermethod known in the art for quantifying reducing sugars.

The hydrolysis product pattern of the non-maltogenic exoamylase isdetermined by incubating 0.7 units of non-maltogenic exoamylase for 15or 300 min. at 50° C. in a test tube with 4 ml of 10 mg/ml waxy maizestarch in the buffer prepared as described above. The reaction isstopped by immersing the test tube for 3 min. in a boiling water bath.

The hydrolysis products are analyzed and quantified by anion exchangeHPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxideand water as eluents, with pulsed amperometric detection and with knownlinear maltooligosaccharides of from glucose to maltoheptaose asstandards. The response factor used for maltooctaose to maltodecaose isthe response factor found for maltoheptaose.

Preferably, the PS4 parent polypeptides (and the PS4 variantpolypeptides) have non-maltogenic exoamylase activity such that if anamount of 0.7 units of said non-maltogenic exoamylase were to incubatedfor 15 minutes at a temperature of 50° C. at pH 6.0 in 4 ml of anaqueous solution of 10 mg preboiled waxy maize starch per ml bufferedsolution containing 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mMcalcium chloride then the enzyme would yield hydrolysis product(s) thatwould consist of one or more linear malto-oligosaccharides of from twoto ten D-glucopyranosyl units and optionally glucose; such that at least60%, at least 70%, at least 80% and/or at least 85% by weight of thesaid hydrolysis products consisting of from two to ten D-glucopyranosylunits and optionally glucose would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units.

For ease of reference, and for the present purposes, the feature ofincubating an amount of 0.7 units of the non-maltogenic exoamylase for15 minutes at a temperature of 50° C. at pH 6.0 in 4 ml of an aqueoussolution of 10 mg preboiled waxy maize starch per ml buffered solutioncontaining 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mM calciumchloride, may be referred to as the “Waxy Maize Starch Incubation Test”.

Thus, alternatively expressed, a preferred non-maltogenic exoamylase ischaracterised as having the ability in the waxy maize starch incubationtest to yield hydrolysis product(s) that would consist of one or morelinear malto-oligosaccharides of from two to ten D-glucopyranosyl unitsand optionally glucose; such that at least 60%, preferably at least 70%,more preferably at least 80% and most preferably at least 85% by weightof the said hydrolysis product(s) would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units.

The hydrolysis products in the waxy maize starch incubation test includeone or more linear malto-oligosaccharides of from two to tenD-glucopyranosyl units and optionally glucose. The hydrolysis productsin the waxy maize starch incubation test may also include otherhydrolytic products. Nevertheless, the % weight amounts of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units arebased on the amount of the hydrolysis product that consists of one ormore linear malto-oligosaccharides of from two to ten D-glucopyranosylunits and optionally glucose. In other words, the % weight amounts oflinear maltooligosaccharides of from three to ten D-glucopyranosyl unitsare not based on the amount of hydrolysis products other than one ormore linear malto-oligosaccharides of from two to ten D-glucopyranosylunits and glucose. The hydrolysis products can be analysed by anysuitable means. For example, the hydrolysis products may be analysed byanion exchange HPLC using a Dionex PA 100 column with pulsedamperometric detection and with, for example, known linearmaltooligosaccharides of from glucose to maltoheptaose as standards.

Preferably, the PS4 variants described here are active during baking andhydrolyse starch during and after the gelatinization of the starchgranules which starts at temperatures of about 55° C. The morethermostable the non-maltogenic exoamylase is the longer time it can beactive and thus the more antistaling effect it will provide. However,during baking above temperatures of about 85° C., enzyme inactivationcan take place. If this happens, the non-maltogenic exoamylase may begradually inactivated so that there is substantially no activity afterthe baking process in the final bread. Therefore preferentially thenon-maltogenic exoamylases suitable for use as described have an optimumtemperature above 50° C. and below 98° C.

Exo-specificity can usefully be measured by determining the ratio oftotal amylase activity to the total endoamylase activity. This ratio isreferred to in this document as a “Exo-specificity index”. In preferredembodiments, an enzyme is considered an exoamylase if it has aexo-specificity index of 20 or more, i.e., its total amylase activity(including exo-amylase activity) is 20 times or more greater than itsendoamylase activity. In highly preferred embodiments, theexo-specificity index of exoamylases is 30 or more, 40 or more, 50 ormore, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. Inhighly preferred embodiments, the exo-specificity index is 150 or more,200 or more, 300 or more, or 400 or more.

The total amylase activity and the endoamylase activity may be measuredby any means known in the art. For example, the total amylase activitymay be measured by assaying the total number of reducing ends releasedfrom a starch substrate. Alternatively, the use of a Betamyl assay isdescribed in further detail in the Examples, and for convenience,amylase activity as assayed in the Examples is described in terms of“Betamyl Units” in the Tables and Figures.

Endoamylase activity may be assayed by use of a Phadebas Kit (Pharmaciaand Upjohn). This makes use of a blue labelled crosslinked starch(labelled with an azo dye); only internal cuts in the starch moleculerelease label, while external cuts do not do so. Release of dye may bemeasured by spectrophotometry. Accordingly, the Phadebas Kit measuresendoamylase activity, and for convenience, the results of such an assay(described in the Examples) are referred to in this document as“Phadebas units”.

In a preferred embodiment, therefore, the exo-specificity index isexpressed in terms of Betamyl Units/Phadebas Units.

Exo-specificity may also be assayed according to the methods describedin the prior art, for example, in Publication Number WO99/50399. Thismeasures exo-specificity by way of a ratio between the endoamylaseactivity to the exoamylase activity. Thus, in a preferred aspect, thePS4 variants described here will have less than 0.5 endoamylase units(EAU) per unit of exoamylase activity. Preferably the non-maltogenicexoamylases which are suitable for use according to the presentinvention have less than 0.05 EAU per unit of exoamylase activity andmore preferably less than 0.01 EAU per unit of exoamylase activity.

The PS4 variant polypeptides, nucleic acids, host cells, expressionvectors, etc, may be used in any application for which an amylase may beused. In particular, they may be used to substitute for anynon-maltogenic exoamylase. They may be used to supplement amylase ornon-maltogenic exoamylase activity, whether alone or in combination withother known amylases or non-maltogenic exoamylases.

The PS4 variant sequences described here may be used in variousapplications in the food industry—such as in bakery and drink products,they may also be used in other applications such as a pharmaceuticalcomposition, or even in the chemical industry. In particular, the PS4variant polypeptides and nucleic acids are useful for various industrialapplications including baking (as disclosed in WO 99/50399) and flourstandardisation (volume enhancement or improvement). They may be used toproduce maltotetraose from starch and other substrates.

The PS4 variant polypeptides may be used to enhance the volume of bakeryproducts such as bread. Thus, food products comprising or treated withPS4 variant polypeptides are expanded in volume when compared toproducts which have not been so treated, or treated with parentpolypeptides. In other words, the food products have a larger volume ofair per volume of food product. Alternatively, or in addition, the foodproducts treated with PS4 variant polypeptides have a lower density, orweight (or mass) per volume ratio. In particularly preferredembodiments, the PS4 variant polypeptides are used to enhance the volumeof bread. Volume enhancement or expansion is beneficial because itreduces the gumminess or starchiness of foods. Light foods are preferredby consumers, and the customer experience is enhanced. In preferredembodiments, the use of PS4 variant polypeptides enhances the volume by10%, 20%, 30% 40%, 50% or more.

The PS4 variant polypeptides and nucleic acids described here may beused as—or in the preparation of—a food. In particular, they may beadded to a food, i.e., as a food additive. In a preferred aspect, thefood is for human consumption. The food may be in the form of a solutionor as a solid—depending on the use and/or the mode of application and/orthe mode of administration.

The PS4 variant polypeptides and nucleic acids may be used as a foodingredient. The PS4 variant polypeptides and nucleic acids disclosedhere may be—or may be added to—food supplements. The PS4 variantpolypeptides and nucleic acids disclosed here may be—or may be addedto—functional foods.

The PS4 variant polypeptides may also be used in the manufacture of afood product or a foodstuff. Typical foodstuffs include dairy products,meat products, poultry products, fish products and dough products. Thedough product may be any processed dough product, including fried, deepfried, roasted, baked, steamed and boiled doughs, such as steamed breadand rice cakes. In highly preferred embodiments, the food product is abakery product.

Preferably, the foodstuff is a bakery product. Typical bakery (baked)products include bread—such as loaves, rolls, buns, pizza bases etc.pastry, pretzels, tortillas, cakes, cookies, biscuits, crackers etc.

The PS4 variant proteins are capable of retarding the staling of starchmedia, such as starch gels. The PS4 variant polypeptides are especiallycapable of retarding the detrimental retrogradation of starch.

Accordingly, the use of PS4 variant polypeptides as described here whenadded to the starch at any stage of its processing into a food product,e.g., before during or after baking into bread can retard or impede orslow down the retrogradation. Such use is described in further detailbelow.

For evaluation of the antistaling effect of the PS4 variant polypeptideshaving non-maltogenic exoamylase activity described here, the crumbfirmness can be measured 1, 3 and 7 days after baking by means of anInstron 4301 Universal Food Texture Analyzer or similar equipment knownin the art.

Another method used traditionally in the art and which is used toevaluate the effect on starch retrogradation of a PS4 variantpolypeptide having non-maltogenic exoamylase activity is based on DSC(differential scanning calorimetry). Here, the melting enthalpy ofretrograded amylopectin in bread crumb or crumb from a model systemdough baked with or without enzymes (control) is measured. The DSCequipment applied in the described examples is a Mettler-Toledo DSC 820run with a temperature gradient of 10° C. per min. from 20 to 95° C. Forpreparation of the samples 10-20 mg of crumb are weighed and transferredinto Mettler-Toledo aluminium pans which then are hermetically sealed.

The model system doughs used in the described examples contain standardwheat flour and optimal amounts of water or buffer with or without thenon-maltogenic PS4 variant exoamylase. They are mixed in a 10 or 50 gBrabender Farinograph for 6 or 7 min., respectively. Samples of thedoughs are placed in glass test tubes (15*0.8 cm) with a lid. These testtubes are subjected to a baking process in a water bath starting with 30min. incubation at 33° C. followed by heating from 33 to 95° C. with agradient of 1.1° C. per min. and finally a 5 min. incubation at 95° C.Subsequently, the tubes are stored in a thermostat at 20° C. prior toDSC analysis.

In preferred embodiments, the PS4 variants described here have a reducedmelting enthalpy, compared to the control. In highly preferredembodiments, the PS4 variants have a 10% or more reduced meltingenthalpy. Preferably, they have a 20% or more, 30%, 40%, 50%, 60%, 70%,80%, 90% or more reduced melting enthalpy when compared to the control.TABLE 2 DSC (J/g) Control 2.29 0.5 D34 1.91 1 D34 1.54 2 D34 1.14

The above Table 2 shows DSC values of model dough systems prepared withdifferent doses of PSac-D34 after 7 days of storage. 0.5, 1 and 2 partsper million (or microgram per gram) of flour are tested.

PS4 variant polypeptides can be used in the preparation of foodproducts, in particular, starch products. The method comprises formingthe starch product by adding a non-maltogenic exoamylase enzyme such asa PS4 variant polypeptide, to a starch medium. If the starch medium is adough, then the dough is prepared by mixing together flour, water, thenon-maltogenic exoamylase which is a PS4 variant polypeptide andoptionally other possible ingredients and additives.

A preferred flour is wheat flour or rye flour or mixtures of wheat andrye flour. However, dough comprising flour derived from other types ofcereals such as for example from rice, maize, barley, and durra are alsocontemplated. Preferably, the starch product is a bakery product. Morepreferably, the starch product is a bread product. Even more preferably,the starch product is a baked farinaceous bread product.

Thus, if the starch product is a baked farinaceous bread product, thenthe process comprises mixing—in any suitable order—flour, water, and aleavening agent under dough forming conditions and further adding a PS4variant polypeptide, optionally in the form of a premix. The leaveningagent may be a chemical leavening agent such as sodium bicarbonate orany strain of Saccharomyces cerevisiae (Baker's Yeast).

The PS4 variant non-maltogenic exoamylase can be added together with anydough ingredient including the water or dough ingredient mixture or withany additive or additive mixture. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

A preferred embodiment is a process for making a bread productcomprising: (a) providing a starch medium; (b) adding to the starchmedium a PS4 variant polypeptide as described in this document; and (c)applying heat to the starch medium during or after step (b) to produce abread product. Another preferred embodiment is a process for making abread product comprising adding to a starch medium a PS4 variantpolypeptide as described.

The non-maltogenic exoamylase PS4 variant polypeptide can be added as aliquid preparation or as a dry pulverulent composition either comprisingthe enzyme as the sole active component or in admixture with one or moreadditional dough ingredients or dough additives.

Another preferred embodiment is the use of such a bread and doughimproving compositions in baking. A further embodiment provides a bakedproduct or dough obtained from the bread improving composition or doughimproving composition. Another embodiment provides a baked product ordough obtained from the use of a bread improving composition or a doughimproving composition.

A dough may be prepared by admixing flour, water, a dough improvingcomposition comprising PS4 variant polypeptide (as described above) andoptionally other ingredients and additives.

The dough improving composition can be added together with any doughingredient including the flour, water or optional other ingredients oradditives. The dough improving composition can be added before the flouror water or optional other ingredients and additives. The doughimproving composition can be added after the flour or water, or optionalother ingredients and additives. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

The dough improving composition can be added as a liquid preparation orin the form of a dry powder composition either comprising thecomposition as the sole active component or in admixture with one ormore other dough ingredients or additive.

The amount of the PS4 variant polypeptide non-maltogenic exoamylase thatis added is normally in an amount which results in the presence in thefinished dough of 50 to 100,000 units per kg of flour, preferably 100 to50,000 units per kg of flour. Preferably, the amount is in the range of200 to 20,000 units per kg of flour.

In the present context, 1 unit of the non-maltogenic exoamylase isdefined as the amount of enzyme which releases hydrolysis productsequivalent to 1 μmol of reducing sugar per min. when incubated at 50degrees C. in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50mM MES, 2 mM calcium chloride, pH 6.0 as described hereinafter.

The dough as described here generally comprises wheat meal or wheatflour and/or other types of meal, flour or starch such as corn flour,corn starch, maize flour, rice flour, rye meal, rye flour, oat flour,oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potatoflour or potato starch. The dough may be fresh, frozen, or part-baked.

The dough may be a leavened dough or a dough to be subjected toleavening. The dough may be leavened in various ways, such as by addingchemical leavening agents, e.g., sodium bicarbonate or by adding aleaven (fermenting dough), but it is preferred to leaven the dough byadding a suitable yeast culture, such as a culture of Saccharomycescerevisiae (baker's yeast), e.g. a commercially available strain of S.cerevisiae.

The dough may comprise fat such as granulated fat or shortening. Thedough may further comprise a further emulsifier such as mono- ordiglycerides, sugar esters of fatty acids, polyglycerol esters of fattyacids, lactic acid esters of monoglycerides, acetic acid esters ofmonoglycerides, polyoxethylene stearates, or lysolecithin.

Another embodiment is a pre-mix comprising flour together with thecombination as described herein. The pre-mix may contain otherdough-improving and/or bread-improving additives, e.g. any of theadditives, including enzymes, mentioned herein.

In order to improve further the properties of the baked product andimpart distinctive qualities to the baked product further doughingredients and/or dough additives may be incorporated into the dough.Typically, such further added components may include dough ingredientssuch as salt, grains, fats and oils, sugar or sweetener, dietary fibres,protein sources such as milk powder, gluten soy or eggs and doughadditives such as emulsifiers, other enzymes, hydrocolloids, flavouringagents, oxidising agents, minerals and vitamins.

The emulsifiers are useful as dough strengtheners and crumb softeners.As dough strengtheners, the emulsifiers can provide tolerance withregard to resting time and tolerance to shock during the proofing.Furthermore, dough strengtheners will improve the tolerance of a givendough to variations in the fermentation time. Most dough strengthenersalso improve on the oven spring which means the increase in volume fromthe proofed to the baked goods. Lastly, dough strengtheners willemulsify any fats present in the recipe mixture.

Suitable emulsifiers include lecithin, polyoxyethylene stearat, mono-and diglycerides of edible fatty acids, acetic acid esters of mono- anddiglycerides of edible fatty acids, lactic acid esters of mono- anddiglycerides of edible fatty acids, citric acid esters of mono- anddiglycerides of edible fatty acids, diacetyl tartaric acid esters ofmono- and diglycerides of edible fatty acids, sucrose esters of ediblefatty acids, sodium stearoyl-2-lactylate, and calciumstearoyl-2-lactylate.

The further dough additive or ingredient can be added together with anydough ingredient including the flour, water or optional otheringredients or additives, or the dough improving composition. Thefurther dough additive or ingredient can be added before the flour,water, optional other ingredients and additives or the dough improvingcomposition. The further dough additive or ingredient can be added afterthe flour, water, optional other ingredients and additives or the doughimproving composition.

The further dough additive or ingredient may conveniently be a liquidpreparation. However, the further dough additive or ingredient may beconveniently in the form of a dry composition.

Preferably the further dough additive or ingredient is at least 1% theweight of the flour component of dough. More preferably, the furtherdough additive or ingredient is at least 2%, preferably at least 3%,preferably at least 4%, preferably at least 5%, preferably at least 6%.If the additive is a fat, then typically the fat may be present in anamount of from 1 to 5%, typically 1 to 3%, more typically about 2%.

Other uses may also be found in attorney docket numbers 674510-2007 andGC806P, all of which are hereby incorporated by reference, including anddrawings, references and Figures.

EXAMPLES Example 1 Cloning of PS4

Pseudomonas saccharophila is grown overnight on LB media and chromosomalDNA is isolated by standard methods (Sambrook J, 1989). A 2190 bpfragment containing 5 the PS4 open reading frame (Zhou et al., 1989) isamplified from P. saccharophila chromosomal DNA by PCR using the primersP1 and P2 (see Table 3). The resulting fragment is used as a template ina nested PCR with primers P3 and P4, amplifying the openreading frame ofPS4 without its signal sequence and introducing a NcoI site at the 5′end of the gene and a BamHI site at the 3′ end. Together with the NcoIsite a codon for a N-terminal Methionine is introduced, allowing forintracellular expression of PS4. The 1605 bp fragment is cloned intopCRBLUNT TOPO (Invitrogen) and the integrity of the construct analysedby sequencing. The E.coli Bacillus shuttle vector pDP66K (Penninga etal., 1996) is modified to allow for expression of the PS4 under controlof the P32 promoter and the ctgase signal sequence. The resultingplasmid, pCSmta is transformed into B. subtilis.

A second expression construct is made in which the starch binding domainof PS4 is removed. In a PCR with primers P3 and P6 (Table 3) on pCSmta,a truncated version of the mta gene is generated. The full length mtagene in pCSmta is exchanged with the truncated version which resulted inthe plasmid pCSmta-SBD.

Example 2 Site Directed Mutagenesis of PS4

Mutations are introduced into the mta gene by 2 methods. Either by a 2step PCR based method, or by a Quick Exchange method (QE). Forconvenience the mta gene is split up in 3 parts; a PvuI-FspI fragment, aFspI-PstI fragment and a PstI-AspI fragment, further on referred to asfragment 1, 2 and 3 respectively.

In the 2 step PCR based method, mutations are introduced using Pfu DNApolymerase (Stratagene). A first PCR is carried out with a mutagenesisprimer (Table 4) for the coding strand plus a primer downstream on thelower strand (either 2R or 3R Table 3).

The reaction product is used as a primer in a second PCR together with aprimer upstream on the coding strand. The product of the last reactionis cloned into pCRBLUNT topo (Invitrogen) and after sequencing thefragment is exchanged with the corresponding fragment in pCSmta.

Using the Quick Exchange method (Stratagene), mutations are introducedusing two complementary primers in a PCR on a plasmid containing the mtagene, or part of the mta gene.

For this purpose a convenient set of plasmids is constructed, comprisingof 3 SDM plasmids and 3 pCSΔ plasmids. The SDM plasmids each bear 1 ofthe fragments of the mta gene as mentioned above, in which the desiredmutation is introduced by QE. After verification by sequencing, thefragments are cloned into the corresponding recipient pCSΔ plasmid. ThepCSΔ plasmids are inactive derivatives from pCSmta. Activity is restoredby cloning the corresponding fragment from the SDM plasmid, enablingeasy screening. TABLE 3 Primers used in cloning the mta gene, andstandard primers used in construction of site directed mutants with the2 step PCR method. Introduced Primer Primer sequence Site P1 5′-ATG ACGAGG TCC TTG TTT TTC P2 5′-CGC TAG TCG TCC ATG TCG P3 5′-GCC ATG GAT CAGGCC GGC AAG NcoI AGC CCG P4 5′-TGG ATC CTC AGA ACG AGC CGC BamHI TGG TP6 5′-GAA TTC AGC CGC CGT CAT TCC EcoRI CGC C 2L 5′-AGA TTT ACG GCA TGTTTC GC 2R 5′-TAG CCG CTA TGG AAG CTG AT 3L 5′-TGA CCT TCG TCG ACA ACC AC3R 5′-GAT AGC TGC TGG TGA CGG TC

TABLE 4 Primers used to introduce site directed mutations in mtaMutation Oligo Sequence Modification Strand Purpose G134RCTGCCGGCCGGCCA + SDM GcGCTTCTGGCG G134R- cgccagaagcgctg − SDMgccggccggcag I157L GACGGTGACCGCTT + SDM CcTgGGCGGCGAGT CG I151L-cgactcgccgccca − SDM ggaagcggtcaccg tc G223A GGCGAGCTGTGGAA + SDMAgccCCTTCTGAAT ATCCG G223A- cggatattcagaag − SDM gggctttccacagc tcgccH307L gaacGGCGGCCAGC + SDM ACctgTGGGCGCTG CAG H307L- ctgcagcgcccaca −SDM ggtgctggccgccg ttc S334P, GTACTGGccgCACA + SDM D343E TGTACGACTGGGGCTACGGCgaaTTCAT C S334P, gatgaattcgccgt − SDM D343E- agccccagtcgtacatgtgcggccagta c

TABLE 5 Features of the SDM and pCSΔ plasmids PlasmidFeatures/construction SDM1 pBlueSK + 480 bp SalI-StuI fragment mta SDM2pBlueSK + 572 bp SacII-PstI fragment mta SDM3 pBlueSK + 471 bp SalI-StuIfragment mta pCSΔ1 FseI site filled in with Klenow ----> frameshift inmta pCSΔ2 FspI-PstI fragment of mta replaced with ‘junk-DNA’ pCSΔ3PstI-AspI fragment of mta replaced with ‘junk-DNA’

Example 3 Multi SDM

The PS4 variants were generated using a QuickChange® Multi Site DirectedMutagenesis Kit (Stratagene) according to the manufactures protocol withsome modifications as described.

Step 1: Mutant Strand Synthesis Reaction (PCR)

-   -   Inoculate 3 ml. LB (22 g/l Lennox L Broth Base,        Sigma)+antibiotics (0,05 μg/ml kanamycin, Sigma) in a 10 ml        Falcon tube    -   Incubate o/n 37° C., ca. 200 rpm.    -   Spin down the cells by centrifugation (5000 rpm/5 min)    -   Poor off the medium    -   Prepare ds-DNA template using QIAGEN Plasmid Mini Purification        Protocol

1. The mutant strand synthesis reaction for thermal cycling was preparedas follow: PCR Mix: 2.5 μl 10X QuickChange ® Multi reaction buffer 0.75μl QuickSolution X μl ${Primers}\quad\begin{pmatrix}{{{primer}\quad{length}\quad 28{–35}\quad{bp}}->{10\quad{pmol}}} \\{{{primer}\quad{length}\quad 24{–27}\quad{bp}}->{7\quad{pmol}}} \\{{{primer}\quad{length}\quad 20{–23}\quad{bp}}->{5\quad{pmol}}}\end{pmatrix}$ 1 ∥l dNTP mix X μl ds-DNA template (200 ng) 1 μlQuickChange ® Multi enzyme blend (2.5 U/μl) (PfuTurbo ® DNA polymerase)X μl dH₂O (to a final volume of 25 μl)

-   -   -   Mix all components by pipetting and briefly spin down the            reaction mixtures.

    -   2. Cycle the reactions using the following parameters:        -   35 cycles of denaturation (96° C./1 min)

primer annealing (62,8° C./1 min)

elongation (65° C./15 min)

then hold at 4° C.

-   -   -   Preheat the lid of the PCR machine to 105° C. and the plate            to 95° C. before the PCR tubes are placed in the machine            (eppendorf thermal cycler).            Step 2: Dpn I Digestion

    -   1. Add 2 μl Dpn I restriction enzyme (10 U/μl) to each        amplification reaction, mix by pipetting and spin down mixture.

    -   2. Incubate at 37° C. for ˜3 hr.        Step 3: Transformation of XL10-Gold® Ultracompetent Cells

    -   1. Thaw XL10-Gold cells on ice. Aliquot 45 μl cells per        mutagenesis reaction to prechilled Falcon tubes.

    -   2. Turn on the waterbath (42° C.) and place a tube with NZY⁺        broth in the bath to preheat.

    -   3. Add 2 μl β-mercaptoethanol mix to each tube. Swirl and tap        gently and incubate 10 min on ice, swirling every 2 min.

    -   4. Add 1,5 μl Dpn I-treated DNA to each aliquot of cells, swirl        to mix and incubate on ice for 30 min.

    -   5. Heat-pulse the tubes in 42° C. waterbath for 30 s and place        on ice for 2 min.

    -   6. Add 0.5 ml preheated NZY⁺ broth to each tube and incubate at        37° C. for 1 hr with shaking at 225-250 rpm.

    -   7. Plate 200 μl of each transformation reaction on LB plates        (33,6 g/l Lennox L Agar, Sigma) containing 1% starch and 0,05        μg/ml kanamycin

8. Incubate the transformation plates at 37° C. overnight. TABLE 6Primer table for pPD77d14: Mutation Oligo Sequence Modification StrandPurpose N33Y, GCGAAGCGCCCTAC 5′ phosphate + MSDM D34N AACTGGTACAAC K71RCCGACGGCGGCAGG 5′ phosphate + MSDM TCCGGCG G87S CAAGAACAGCCGCT 5′phosphate + MSDM ACGGCAGCGAC G121D GACATGAACCGGGA 5′ phosphate + MSDMCTACCCGGACAAG G134R CTGCCGGCCGGCCA 5′ phosphate + MSDM GcGCTTCTGGCGA141P CGCAACGACTGCGC 5′ phosphate + MSDM CGACCCGGG I157L GACGGTGACCGCTT5′ phosphate + MSDM CcTgGGCGGCGAGT CG L178F, CGCGACGAGTTTAC 5′phosphate + MSDM A179T CAACCTGCG G223A GGCGAGCTGTGGAA 5′ phosphate +MSDM AgccCCTTCTGAAT ATCCG H307L gaacGGCGGCCAGC 5′ phosphate + MSDMACctgTGGGCGCTG CAG S334P, GTACTGGccgCACA 5′ phosphate + MSDM D343ETGTACGACTGGGGC TACGGCgaaTTCAT C

TABLE 7 Primer table for pPD77d20: Mutation Oligo Sequence ModificationStrand Purpose N33Y, GCGAAGCGCCCTAC 5′ phosphate + MSDM D34NAACTGGTACAAC K71R CCGACGGCGGCAGG 5′ phosphate + MSDM TCCGGCG G121DCACATGAACCGCGA 5′ phosphate + MSDM CTACCCGGACAAG G134R CTGCCGGCCGGCCA 5′phosphate + MSDM GcGCTTCTGGCG A141P CGCAACGACTGCGC 5′ phosphate + MSDMCGACCCGGG I157L GACGGTGACCGCTT 5′ phosphate + MSDM CcTgGGCGGCGAGT CGL178F, CGCGACGAGTTTAC 5′ phosphate + MSDM A179T CAACCTGCG G223AGGCGAGCTGTGGAA 5′ phosphate + MSDM AgccCCTTCTGAAT ATCCG H307LgaacGGCGGCCAGC 5′ phosphate + MSDM ACctgTGGGCGCTG CAG S334P,GTACTGGccgCACA 5′ phosphate + MSDM D343E TGTACGACTGGGGC TACGGCgaaTTCAT C

TABLE 8 Primer table for pPD77d34: Mutation Oligo Sequence ModificationStrand Purpose N33Y, GCGAAGCGCCCTAC 5′ phosphate + MSDM D34NAACTGGTACAAC G121D CACATGAACCGCGA 5′ phosphate + MSDM CTACCCGGACAAGG134R CTGCCGGCCGGCCA 5′ phosphate + MSDM GcGCTTCTGGCG A141PCGCAACGACTGCGC 5′ phosphate + MSDM CGACCCGGG I157L GACGGTGACCGCTT 5′phosphate + MSDM CcTgGGCGGCGAGT CG L178F, CGCGACGAGTTTAC 5′ phosphate +MSDM A179T CAACCTGCG G223A GGCGAGCTGTGGAA 5′ phosphate + MSDMAgccCCTTCTGAAT ATCCG H307L gaacGGCGGCCAGC 5′ phosphate + MSDMACctgTGGGCGCTG CAG S334P GTACTGGccgCACA 5′ phosphate + MSDMTGTACGACTGGGGC TACGGCVector system based on pPD77

The vector system used for pPD77 is based on pCRbluntTOPOII(invitrogen). The zeocin resistance cassette has been removed by pmlI,393 bp fragment removed. The expression cassette from the pCC vector(P32-ssCGTase-PS4-tt) has then been inserted into the vector.

Ligation of PS4 Variant into pCCMini

The plasmid which contain the relevant mutations (created by MSDM) iscut with restriction enzyme Nco 1 and Hind III (Biolabs):

-   -   3 μg plasmid DNA, X μl 10× buffer 2, 10 units Nco1, 20 units        HindIII,

Incubation 2 h at 37° C.

Run digestion on a 1% agarose gel. Fragments sized 1293 bp (PS4 gene) iscut out of the gel and purified using Qiagen gel purification kit.

The vector pCCMini is then cut with restriction enzymes, Nco 1 and HindIII, and the digestion is then run on a 1% agarose gel. The fragmentsized 3569 bp is cut out of the gel and purified using Qiagen gelpurification kit.

Ligation: Use Rapid DNA ligation kit (Roche)

Use the double amount of insert compared to vector

-   -   e.g. 2 μl insert (PS4 gene)    -   1 μl vector    -   5 μl T4 DNA ligation buffer 2xconc    -   1 μl dH₂O    -   1 μl T4 DNA ligase

Ligate 5 min/RT

Transform the ligation into One Shot TOPO competent cells according tomanufactures protocol (Invitrogen). Use 5 μl ligation pr.transformation.

Plate 50 μl transformationsmix onto LB plates (33,6 g/l Lennox L Agar,Sigma) containing 1% starch and 0,05 μg/ml kanamycin. Vectors containinginsert (PS4 variants) can be recognised by halo formation on the starchplates.

Example 4 Transformation into Bacillus subtilis (ProtoplastTransformation)

Bacillus subtilis (strain DB104A; Smith et al. 1988; Gene 70, 351-361)is transformed with the mutated pCS-plasmids according to the followingprotocol.

A. Media for protoplasting and transformation 2 × SMM per litre: 342 gsucrose (1 M); 4.72 g sodium maleate (0.04 M); 8:12 g MgCl₂, 6H₂0 (0.04M); pH 6.5 with concentrated NaOH. Distribute in 50-ml portions andautoclave for 10 min. 4 × YT 2 g Yeast extract + 3.2 g Tryptone + 0.5 gNaCl (½ NaCl) per 100 ml. mix equal volumes of 2 × SMM and 4 × YT. SMMP10 g polyethyleneglycol 6000 (BDH) or 8000 (Sigma) PEG in 25 ml 1 × SMM(autoclave for 10 min.).

B. Media for Plating/Regeneration agar 4% Difco minimal agar. Autoclavefor 15 min. sodium succinate 270 g/l (1 M), pH 7.3 with HCl. Autoclavefor 15 min. phosphate buffer 3.5 g K₂HPO₄ + 1.5 g KH₂PO₄ per 100 ml.Autoclave for 15 min. MgCl₂ 20.3 g MgCl₂, 6H₂O per 100 ml (1 M).casamino acids 5% (w/v) solution. Autoclave for 15 min. yeast extract 10g per 100 ml, autoclave for 15 min. glucose 20% (w/v) solution.Autoclave for 10 min.

DM3 regeneration medium: mix at 60 C (waterbath; 500-ml bottle):

-   -   250 ml sodium succinate    -   50 ml casamino acids    -   25 ml yeast extract    -   50 ml phosphate buffer    -   15 ml glucose    -   10 ml MgCl₂    -   100 ml molten agar

Add appropriate antibiotics: chloramphenicol and tetracycline, 5 μg/ml;erythromycin, 1 ug/ml. Selection on kanamycin is problematic in DM3medium: concentrations of 250 ug/ml may be required.

C. Preparation of Protoplasts

1. Use detergent-free plastic or glassware throughout.

2. Inoculate 10 ml of 2×YT medium in a 100-ml flask from a singlecolony. Grow an overnight culture at 25-30 C in a shaker (200 rev/min).

3. Dilute the overnight culture 20 fold into 100 ml of fresh 2×YT medium(250-ml flask) and grow until OD₆₀₀=0.4-0.5 (approx. 2 h) at 37C in ashaker (200-250 rev/min).

4. Harvest the cells by centrifugation (9000 g, 20 min, 4 C).

5. Remove the supernatant with pipette and resuspend the cells in 5 mlof SMMP+5 mg lysozyme, sterile filtered.

6. Incubate at 37 C in a waterbath shaker (100 rev/min).

After 30 min and thereafter at 15 min intervals, examine 25 ul samplesby microscopy. Continue incubation until 99% of the cells areprotoplasted (globular appearance). Harvest the protoplasts bycentrifugation (4000 g, 20 min, RT) and pipette off the supernatant.Resuspend the pellet gently in 1-2 ml of SMMP.

The protoplasts are now ready for use. (Portions (e.g. 0.15 ml) can befrozen at −80 C for future use (glycerol addition is not required).Although this may result in some reduction of transformability, 106transformants per ug of DNA can be obtained with frozen protoplasts).

D. Transformation

1. Transfer 450 ul of PEG to a microtube.

2. Mix 1-10 ul of DNA (0.2 ug) with 150 ul of protoplasts and add themixture to the microtube with PEG. Mix immediately, but gently.

3. Leave for 2 min at RT, and then add 1.5 ml of SMMP and mix.

4. Harvest protoplasts by microfuging (10 min, 13.000 rev/min (10-12.000g)) and pour off the supernatant. Remove the remaining droplets with atissue.

Add 300 ul of SMMP (do not vortex) and incubate for 60-90 min at 37 C ina waterbath shaker (100 rev/min) to allow for expression of antibioticresistance markers. (The protoplasts become sufficiently resuspendedthrough the shaking action of the waterbath.). Make appropriatedilutions in 1×SSM and plate 0.1 ml on DM3 plates

Example 5 Fermentation of PS4 Variants in Shake Flasks

The shake flask substrate is prepared as follows: Ingredient %(w/v)Water — Yeast extract 2 Soy Flour 2 NaCl 0.5 Dipotassium phosphate 0.5Antifoam agent 0.05

The substrate is adjusted to pH 6.8 with 4N sulfuric acid or sodiumhydroxide before autoclaving. 100 ml of substrate is placed in a 500 mlflask with one baffle and autoclaved for 30 minutes. Subsequently, 6 mlof sterile dextrose syrup is added. The dextrose syrup is prepared bymixing one volume of 50% w/v dextrose with one volume of water followedby autoclaving for 20 minutes.

The shake flask are inoculated with the variants and incubated for 24hours at 35° C./180 rpm in an incubator. After incubation cells areseparate from broth by centrifugation (10.000×g in 10 minutes) andfinally, the supernatant is made cell free by microfiltration at 0,21μm. The cell free supernatant is used for assays and application tests.

Example 6 Amylase Assays

Betamyl Assay

One Betamyl unit is defined as activity degrading 0,0351 mmole per 1min. of PNP-coupled maltopentaose so that 0,0351 mmole PNP per 1 min.can be released by excess a-glucosidase in the assay mix. The assay mixcontains 50 ul 50 mM Na-citrate, 5 mM CaCl2, pH 6,5 with 25 ul enzymesample and 25 ul Betamyl substrate (Glc5-PNP and a-glucosidase) fromMegazyme, Ireland (1 vial dissolved in 10 ml water). The assay mix isincubated for 30 min. at 40C and then stopped by adding 150 ul 4% Tris.Absorbance at 420 nm is measured using an ELISA-reader and the Betamylactivity is calculate based on Activity=A420*d in Betamyl units/ml ofenzyme sample assayed.

Endo-amylase Assay

The endo-amylase assay is identical to the Phadebas assay run accordingto manufacturer (Pharmacia & Upjohn Diagnostics AB).

Exo-specificity

The ratio of exo-amylase activity to Phadebas activity was used toevaluate exo-specificity.

Specific Activity

For the PSac-D14, PSac-D20 and PSac-D34 variants we find an averagespecific activity of 10 Betamyl units per microgram of purified proteinmeasured according to Bradford (1976; Anal. Biochem. 72, 248). Thisspecific activity is used for based on activity to calculate the dosagesused in the application trials.

Example 7 Half-life Determination

t½ is defined as the time (in minutes) during which half the enzymeactivity is inactivated under defined heat conditions. In order todetermine the half-life of the enzyme, the sample is heated for 1-10minutes at constant temperatures of 60° C. to 90° C. The half life iscalculated based on the residual Betamyl assay.

Procedure: In an Eppendorf vial, 1000 μl buffer is preheated for atleast 10 minutes at 60° C. or higher. The heat treatment of the sampleis started addition of 100 μl of the sample to the preheated bufferunder continuous mixing (800 rpm) of the Eppendorf vial in an heatincubator (Termomixer comfort from Eppendorf). After 0, 2, 4, 6, 8 and 9minutes of incubation, the treatment is stopped by transferring 45 μl ofthe sample to 1000 μl of the buffer equilibrated at 20° C. andincubating for one minute at 1500 rpm and at 20° C. The residualactivity is measured with the Betamyl assay.

Calculation: Calculation of t½ is based on the slope of log 10 (thebase-10 logarithm) of the residual Betamyl activity versus theincubation time. t½ is calculated as Slope/0.301=t½.

Example 8. Results TABLE 9 Biochemical properties of the PSac-variantscompared to wild-type PSac- T½- T½- Betamyl/ Variant 75 80 phadebasMutations Psac-cc1 <0.5 40 None Psac-D3 9.3 3 43 G134R A141P I157L G223AH307L S334P D343E N33Y D34N K71R L178F A179T Psac-D14 9.3 2.7 65 G134RA141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121DPsac-D20 7.1 2.7 86 G87S G134R A141P I157L G223A H307L S334P D343E N33YD34N K71R L178F A179T G121D Psac-D34 8.4 2.9 100 G134R A141P I157L G223AH307L S334P N33Y D34N L178F A179T G121Dcc1

TABLE 10 pPD77 3.6 1.3 20 G134R, A141P I157L G223A H307L S334P D343EpPD77d1 10.3  4 20 G134R, A141P I157L G223A H307L S334P D343E N33Y D34NK71R G158D L178F A179T pPD77d2 4.2 21 G134R, A141P I157L G223A H307LS334P D343E G158D pPD77d3 8.9 3.1 35 G134R, A141P I157L G223A H307LS334P D343E N33Y D34N K71R L178F A179T pPD77d5 10¹   43 G134R, A141PI157L G223A H307L S334P D343E N33Y D34N G158D pPD77d6 4.2 14 G134R,A141P I157L G223A H307L S334P D343E N33Y D34N K71R G158D L178F A179TpPD77d10 3.7 61 G134R, A141P I157L G223A H307L S334P D343E G121DpPD77d11 2.8 57 G134R A141P I157L G223A H307L S334P D343E N33Y G121DpPD77d12 3.8 53 G134R A141P I157L G223A H307L S334P D343E N33Y

TABLE 11 pPD77d3 8.9 3.1 35 G134R, A141P I157L G223A H307L S334P D343EN33Y D34N K71R L178F A179T pPD77d14 9.3 2.8 66 G134R, A141P I157L G223AH307L S334P D343E N33Y D34N K71R L178F A179T G87S G121D S214N T375ApPD77d17 1.3 0.5 86 G134R, A141P I157L G223A H307L S334P D343E N33Y D34NK71R L178F A179T G121D Y171S G188A N138D pPD77d20 7.1 2.7 81 G134R,A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D

TABLE 12 pPD77d14 9.3 2.8 66 G87S G121D G134R, A141P I157L G223A H307LS334P D343E N33Y D34N K71R L178F A179T pPD77d25 2.4 71 G87S G121D G134R,A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G188A

TABLE 12 pPD77d3 8.9 3.1 35 G134R, A141P I157L G223A H307L S334P D343EN33Y D34N K71R L178F A179T pPD77d31 2.5 53 G134R, A141P I157L G223AH307L S334P D343E N33Y D34N K71R L178F A179T Y33N N34D E343D pPD77d322.5 52 G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178FA179T Y33N N34D R71K G87S G121D E343D pPD77d33 7.1 3.0 51 G134R, A141PI157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T R71K E343DpPD77d34 8.4 2.9 67 G134R, A141P I157L G223A H307L S334P D343E N33Y D34NK71R L178F A179T Y33N N34D R71K G87S G121D E343D pPD77d38 7.9 2.5 77G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179TY33N N34D R71K G121D E343D pPD77d39 7.5 2.6 42 G134R, A141P I157L G223AH307L S334P D343E N33Y D34N K71R L178F A179T E343D pPD77d40 10.26 3.1 63G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179TG121D E343D

TABLE 13 pPD77d14 9.3 2.8 66 G87S G121D G134R, A141P I157L G223A H307LS334P D343E N33Y D34N K71R L178F A179T pPD77d35 0 G87S G121D G134R,A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T Y33N N34DR71K E343D pPD77d36 2.8 77 G87S G121D G134R, A141P I157L G223A H307LS334P D343E N33Y D34N K71R L178F A179T Y33N N34D E343D

TABLE 14 pPD77d20 7.1 2.7 81 G134R, A141P I157L G223A H307L S334P D343EN33Y D34N K71R L178F A179T G121D pPD77d37 7.8 2.9 78 G134R, A141P I157LG223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D Y33N N34D E343D

TABLE 15 Activity Identifier (U/ml) T½ (65B) T½ (70B) T½ (72B) T½ (75B)T½ (80B) Mut_OverV SSM53 123 7.5 4.2 0.7 G87S, V113I, G134R, A141P, F6I157L, Y198F, G223A, V290I, H307L, S334P, D343E MS18 101 11.9 I113F,A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E MC033 172 12.94.2 2.6 1.0 A99V, V113I, A141P, I157L, Y198F, G223A, V290I, H307L,S334P, D343E MC037 138 0.1 V113I, I157L, Y198F, G223A, V290I, H307L,S334P, D343E MC032 177 41.5 7.4 4.2 1.1 V113I, G134R, A141P, I157L,Y198F, G223A, V290I, H307L, S334P, D343E MC031 282 18.6 4.7 4.8 1.3A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E MC028 118 9.3 4.42.7 A199V, D343E, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334PMC027 87 11.4 4.3 2.1 0.4 V113I, A141P, I157L, Y198F, G223A, V290I,S334P, D343E MC045 73 1.3 V113I, G121D, G134R, A141P, I157L, Y198F,G223A, V290I, H307L, S334P, D343E MC021 41 4.9 V113I, A141P, Y198F,G223A, V290I, H307I, S334P, D343E MC051 86 9.2 5.2 1.3 V113I, G121D,G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E MC023 1708.1 4.6 2.9 1.0 V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P,D343E MC010 20 V113A, A141P, Y198W, G223A, V290I MC020 141 3.7 0.8V113I, A141P, Y198F, G223A, V290I, H307L MC019 145 5.2 1.6 V113I, A141P,Y198F, G223A, V290I, S334P, D343E MC018 73 3.3 V113I, A141P, Y198F,G223A, A268P, V290I, S399P MC013 104 2.9 V113I, A141P, Y198F, G223A,V290I, S399P MC011 50 7.5 V113I, A141P, Y198W, G223A, V290I MC008 2763.5 V113I, A141P, Y198F, G223A, V290I MC005 20 V113L, A141P, Y198F,G223A, V290I MC004 24 V113A, A141P, Y198F, G223A, V290I MC002 213 Y198F,G223A, V290I MC001 82 3.4 Y198W, G223A, V290I MC022 79 8.5 1.2 V113I,A141P, I157L, Y198F, G223A, V290I MP11 V290I MP04 51 V290I S298 90 1.1V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343ES297 69 1.6 V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,I315V, S334P, D343E S294 121 5.7 2.1 D34N, V113I, G134R, A141P, I157L,Y198F, G223A, V290I, H307L, S334P, D343E S263 81 5.3 2.1 V113I, G134R,A141P, I157L, G188S, Y198F, G223A, V290I, H307L, S334P, D343E S260 585.8 1.8 D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,G313G, S334P, D343E S259 106 5.9 1.8 V113I, G134R, A141P, I157L, Y198F,S214G, G223A, V290I, H307L, S334P, D343E S290 141 5.5 1.5 V113I, G134R,A141P, I157L, A179V, Y198F, G223A, V290I, H307L, S334P, D343E S286 15012.0 6.6 1.8 V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I,H307L, G313G, S334P, D343E SSM53 121 3.1 G87S, V113I, G134R, A141P, E9I157L, Y198F, G223A, V290I, H307L, S334P, D343E S242 83 7.4 3.2 1.1V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P,D343E SSM884 31 6.9 4.1 V113I, G134R, A141P, F6 I157L, Y198F, G223A,V290I, H307L, S334P, D343E, G400G, A405S SSM884 31 6.0 3.7 V113I, G134R,A141P, E6 I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405E SSM88427 6.6 4.0 V113I, G134R, A141P, E4 I157L, Y198F, G223A, V290I, H307L,S334P, D343E, A398A, A405F SSM884 92 8.3 4.0 V113I, G134R, A141P, A11I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405V S220 129 17.0 5.43.2 1.2 A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E S241 1669.0 5.7 1.8 V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L,S334P, D343E S240 115 10.4 3.4 V113I, G134R, A141P, I157L, Y198F, G223A,V290I, H307L, S334P, D343E S239 71 9.7 4.6 1.6 K71R, V113I, G134R,A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E S237 72 10.2 4.41.3 K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L,S334P, D343E S231 55 9.7 4.5 1.6 D34G, V113I, G134R, A141P, I157L,Y198F, G223A, V290I, H307L, S334P, D343E S219 99 16.7 5.2 2.6 1.1 G4D,V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E S227 1079.7 5.5 2.0 A141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F,G188A S226 107 3.1 A141P, G134R, G223A, H307L, I157L, V113I, V290I,Y198F, S183N?

Example 9 Model System Baking Tests

The doughs are made in the Farinograph at 30.0° C. 10.00 g reformedflour is weighed out and added in the Farinograph; after 1 min. mixingthe reference/sample (reference=buffer or water, sample=enzyme+buffer orwater) is added with a sterile pipette through the holes of the kneadingvat. After 30 sec. the flour is scraped off the edges—also through theholes of the kneading vat. The sample is kneaded for 7 min.

A test with buffer or water is performed on the Farinograph before thefinal reference is run. FU should be 400 on the reference, if it is not,this should be adjusted with, for example, the quantity of liquid. Thereference/sample is removed with a spatula and placed in the hand (witha disposable glove on it), before it is filled into small glass tubes(of approx. 4.5 cm's length) that are put in NMR tubes and corked up. 7tubes per dough are made.

When all the samples have been prepared, the tubes are placed in a(programmable) water bath at 33° C. (without corks) for 25 min. andhereafter the water bath is set to stay for 5 min. at 33° C., then toheated to 98° C. over 56 min. (1.1° C. per minute) and finally to stayfor 5 min. at 96° C.

The tubes are stored at 20.0° C. in a thermo cupboard. The solid contentof the crumb was measured by proton NMR using a Bruker NMS 120 MinispecNMR analyser at day 1, 3. and 7 as shown for crumb samples prepared with0, 05, 1 and 2 ppm PSacD34 in FIG. 2. The lower increase in solidcontent over time represents the reduction in amylopectinretrogradation. After 7 days of storage at 20.0° C. in a thermo cupboard10-20 mg samples of crumb weighed out and placed in 40 μl aluminiumstandard DSC capsules and kept at 20° C.

The capsules are used for Differential Scanning Calorimetry on a MettlerToledo DSC 820 instrument. As parameters are used a heating cycle of20-95° C. with 10° C. per min. heating and Gas/flow: N₂/80 ml per min.The results are analysed and the enthalpy for melting of retrogradedamylopectin is calculated in J/g.

Example 10 Antistaling Effects

Model bread crumbs are prepared and measured according to Example 8. Asshown in Table 2, PS4 variants show a strong reduction of theamylopectin retrogradation after baking as measured by DifferentialScanning Calorimetry in comparison to the control. The PS4 variantsshows a clear dosage effect.

Example 11 Firmness Effects in Baking Trials

Baking trials were carried out with a standard white bread sponge anddough recipe for US toast. The sponge dough is prepared from 1600 g offlour “All Purpose Classic” from Sisco Mills, USA”, 950 g of water, 40 gof soy bean oil and 32 g of dry yeast. The sponge is mixed for 1 min. atlow speed and subsequently 3 min. at speed 2 on a Hobart spiral mixer.The sponge is subsequently fermented for 2,5 hours at 35° C., 85% RHfollowed by 0,5 hour at 5° C.

Thereafter 400 g of flour, 4 g of dry yeast, 40 g of salt, 2,4 g ofcalcium propionate, 240 g of high fructose corn syrup (Isosweet), 5 g ofthe emulsifier PANODAN 205, 5 g of enzyme active soy flour, 30 g ofnon-active soy flour, 220 g of water and 30 g of a solution of ascorbicacid (prepared from 4 g ascorbic acid solubilised in 500 g of water) areadded to the sponge. The resulting dough is mixed for 1 min. at lowspeed and then 6 min. on speed 2 on a Diosna mixer. Thereafter the doughis rested for 5 min. at ambient temperature, and then 550 g dough piecesare scaled, rested for 5 min. and then sheeted on Glimek sheeter withthe settings 1:4, 2:4, 3:15, 4:12 and 10 on each side and transferred toa baking form. After 60 min. proofing at 43° C. at 90% RH the doughs arebaked for 29 min. at 218° C.

Firmness and resilience were measured with a TA-XT 2 texture analyser.The Softness, cohesiveness and resilience is determined by analysingbread slices by Texture Profile Analysis using a Texture Analyser FromStable Micro Systems, UK. The following settings were used:

Pre Test Speed: 2 mm/s

Test Speed: 2 mm/s

Post Test Speed: 10 mm/s

Rupture Test Distance: 1%

Distance: 40%

Force: 0.098 N

Time: 5.00 sec

Count: 5

Load Cell: 5 kg

Trigger Type: Auto-0.01 N

Results are shown in FIGS. 3 and 4.

Example 12 Control of Volume of Danish Rolls

Danish Rolls are prepared from a dough based on 2000 g Danish reformflour (from Cerealia), 120 g compressed yeast, 32 g salt, and 32 gsucrose. Water is added to the dough according to prior wateroptimisation.

The dough is mixed on a Diosna mixer (2 min. at low speed and 5 min. athigh speed). The dough temperature after mixing is kept at 26° C. 1350 gdough is scaled and rested for 10 min. in a heating cabinet at 30° C.The rolls are moulded on a Fortuna molder and proofed for 45 min. at 34°C. and at 85% relative humidity. Subsequently the rolls are baked in aBago 2 oven for 18 min. at 250° C. with steam in the first 13 seconds.After baking the rolls are cooled for 25 min. before weighing andmeasuring of volume.

The rolls are evaluated regarding crust appearance, crumb homogeneity,capping of the crust, ausbund and specific volume (measuring the volumewith the rape seed displacement method).

Based on these criteria it is found that the PS4 variants increase thespecific volume and improve the quality parameters of Danish rolls. ThusPS4 variants are able to control the volume of baked products.

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

1. An exo-specific non-maltogenic exoamylase comprising an amino acidsequence at least 75% identical with SEQ ID Nos. 1, 5, 7 or 11 and atleast one substitution selected from the group consisting of 4, 33, 34,70, 71, 87, 99, 108, 113, 121, 134, 141, 157, 158, 171, 178, 179, 188,198, 199, 223, 290, 307, 315, 334, 343, 399, and
 405. 2. The exoamylaseof claim 1, wherein said amino acid sequence is at least 75% identicalwith SEQ ID NO.:1.
 3. The exoamylase of claim 1, wherein said amino acidsequence is at least 75% identical with SEQ ID NO.:5.
 4. The exoamylaseof claim 1, wherein said amino acid sequence is at least 75% identicalwith SEQ ID NO.:7.
 5. The exoamylase of claim 1, wherein said amino acidsequence is at least 75% identical with SEQ ID NO.:11.
 6. The exoamylaseof claim 1, wherein said amino acid sequence comprises an amino acidsequence at least 75% identical with SEQ ID Nos. 2-4b or 8-10.
 7. Theexoamylase of claim 1, wherein said at least one substitution isselected from G4D, N33Y, D34N, G70D, K71R, G87S, A99V, K108R, V113I,G121D, G134R, A141P, I157L, G158D, Y171S, L178F, A179T, G188A, Y198F,Y198F, Y198L, A199v, G223A, V290I, H307L, I315V, S334P, D343E, S399P,A405F and A405E.
 8. The exoamylase of claim 1 wherein said at least oneposition is selected from 33, 34, 71, 87, 121, 134, 141, 157, 178, 179,223, 207, 334 and
 343. 9. The exo amylase of claim 8, wherein said atleast one substitution is selected from N33Y, D34N, K71R, G87S, G121DG134R, A141P, I157L, L178F, A179T, G223A, H307L, S334P, and D343E. 10.The exo amylase of claim 8, further comprising at least one additionalsubstitution at a position selected from 108, 158, 171 and
 188. 11. Theexo-amylase of claim 10, wherein said at least one additionalsubstitution is selected from K108R, G158D, Y171S, and G188A.
 12. Theexo-amylase of claim 1, wherein said at least one substitution comprisesa combination selected from the following: G134R, A141P I157L G223AH307L S334P D343E G121D; G134R A141P I157L G223A H307L S334P D343E N33YG121D; G134R A141P I157L G223A H307L S334P D343E N33Y; G134R, A141PI157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G87S G121DS214N T375A; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71RL178F A179T G121D Y171S G188A N138D; G134R, A141P I157L G223A H307LS334P D343E N33Y D34N K71R L178F A179T G121D; G87S G121D G134R, A141PI157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T; G87S G121DG134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179TG188A; G134R, A141P I157L G223A H307L S334P K71R L178F A179T; G134R,A141P I157L G223A H307L S334P L178F A179T; G134R, A141P I157L G223AH307L S334P N33Y D34N L178F A179T; G134R, A141P I157L G223A H307L S334PL178F A179T G87S G121D; G134R, A141P I157L G223A H307L S334P L178F A179TG121D; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178FA179T G121D E343D; G87S G121D G134R, A141P I157L G223A H307L S334P D343EN33Y D34N K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307LS334P D343E N33Y D34N K71R L178F A179T Y33N N34D E343D; G134R, A141PI157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D; G134R,A141P I157L G223A H307L S334P K71R L178F A179T G121D; G87S, V113I,G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; 113F,A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A99V, V113I,A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, I157L,Y198F, G223A, V290I, H307L, S334P, D343E V113I, G134R, A141 P, I157L,Y198F, G223A, V290I, H307L, S334P, D343E; A141P, I157L, Y198F, G223A,V290I, H307L, S334P, D343E; A199V, D343E, V131I, A141P, I157L, Y198F,G223A, V290I, H307L, S334P; V113I, A141 P, I157L, Y198F, G223A, V290I,S334P, D343E; V113I, G121D, G134R, A141 P, I157L, Y198F, G223A, V290I,H307L, S334P, D343E; V113I, G121D, G134R, A141 P, I157L, Y198F, G223A,V290I, H307L, S334P, D343E; V113I, A141P, I157L, Y198F, G223A, V290I,H307L, S334P, D343E; V113I, A141P, Y198F, G223A, V290I, H307L; V113I,A141P, Y198F, G223A, V290I, S334P, D343E; V113I, A141P, Y198F, G223A,A268P, V290I, S399P V113I, A141 P, Y198F, G223A, V290I, S399P; V113I,A141P, Y198W, G223A, V290I; V113I, A141P, Y198F, G223A, V290I; Y198F,G223A, V290I; Y198W, G223A, V290I; V113, A141P, I157L, Y198F, G223A,V290I; V113M; V113A; V113I, G134R, A141P, I157L, Y198F, G223A, V290I,H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I,H307L, I315V, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F,G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, G188S,Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R, A141P,I157L, L178L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; D34N,V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P,D343E; V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L,S334P, D343E; V113I, G134R, A141P, I157L, I170I, Y198F, G223A, V290I,H307L, G313G, S334P, D343E V113I, G134R, A141P, I157L, A179V, Y198F,G223A, V290I, H307L, G313G, S334P, D343E; G87S, V113I, G134R, A141P,I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P,I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405E; V113I, G134R,A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405V; A141P,I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P,I157L, Y198L, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P,I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R,A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K108R, V113I,G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; D34G,V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; andA141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F, G188A;
 12. Theexo amylase of claim 3, wherein said at least one substitution comprisesa combination selected from the following: G134R, A141P, I157L, G223A,H307L and S334P; G121D, G134R, A141P, I157L, G223A, H307L and S334;G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P; G134R, A141P,I157L, L178F, A179T, G223A, H307L and S334P; G87S, G121D, G134R, A141P,I157L, L178F, A179T, G223A, H307L and S334P; G121D, G134R, A141P, I157L,L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G134R, A141P, I157L,L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G121D, G134R, A141P,I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S,G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. 13.The exo amylase of claim 3, wherein said at least one substitutioncomprises a combination selected from the following: N33Y, D34N, G134R,A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G87S,G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; andN33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307Land S334P
 14. The exo amylase of claim 1, wherein said amylase comprisesan amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:4a, SEQ ID NO:4b, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10.
 15. The exo-amylase of claim 1, wherein said exo-amylase isderived from a Pseudomonas sp.
 16. The exo-amylase of claim 15, whereinsaid Pseudomonas sp. is selected from Pseudomonas saccharophilia andPseudomonas stutzeri.
 17. A nucleic acid sequence at least 75% identicalto the nucleic acid sequence encoding the exo-amylase of claim
 1. 18. Avector comprising the nucleic acid sequence of claim
 17. 19. A host cellcomprising the nucleic acid sequence of claim 18.